Nucleic acid amplification reaction apparatus

ABSTRACT

Apparatus and method for performing a nucleic acid amplification reaction and preferably a polymerase chain reaction (PCR) in a reaction mixture in at least one capillary tube. Several different embodiments are disclosed. One embodiment cycles a sample through a capillary tube loop passing through two thermostatted fluid baths. Another embodiment has the capillary tube routed alternatingly between two heat exchangers to that the sample makes only one pass through the tube. Other embodiments maintain the heat exchangers stationary and translate the samples between them. Still further embodiments maintain the samples stationary and either automatically translate or rotate the heat exchangers past the samples contained within the capillary tubes to perform the thermal cycles necessary for the amplification reaction.

This is a divisional of application Ser. No. 08/299,033 filed on Aug.31, 1994; now U.S. Pat. No. 5,720,923; which is a Continuation of Ser.No. 08/098,711 filed on Jul. 28, 1993, abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to amplifying nucleic acids by thermal cyclingand, more particularly, to automated machines for performingamplification reactions such as a polymerase chain reaction (PCR).

2. Description of the Related Art

DNA (Deoxyribonucleic acid) may be amplified by thermally cycling aspecially constituted liquid reaction mixture according to protocol suchas a polymerase chain reaction (PCR) protocol which includes severalincubations at different temperatures. The reaction mixture is comprisedof various components such as the DNA to be amplified (the target) andat least two oligonucleotide primers selected in a predetermined way soas to be complementary to a portion of the target DNA. The reactionmixture also includes various buffers, enzymes, and deoxyribonucleotidetriphosphates, such as DATP, dCTP, dGTP, and dTTP. The duplex DNAmolecule is denatured into two complementary single strands. The primersthen anneal to the strands, and, in PCR, nucleoside monophosphateresidues are then linked to the primers in the presence of an enzymesuch as a thermostable DNA polymerase to create a primer extensionproduct. After primer extension, twice as many duplex DNA moleculesexist. This process is repeated, each time approximately doubling theamount of DNA present. The result is an exponential increase in theconcentration of target DNA, known as "amplification" of the target DNA.

The polymerase chain reaction (PCR) has proven to be a phenomenalsuccess for genetic analysis, largely because it is simple and veryversatile, and requires relatively low cost instrumentation. A key tothis success is the concept of thermal cycling: alternating steps ofmelting DNA, annealing short primers to the resulting single strands,and extending those primers to make new copies of the double strandedDNA.

The methodology of the polymerase chain reaction is more fully describedin U.S. Pat. Nos. 4,683,202 and 4,683,195 which are hereby incorporatedby reference.

The polymerase chain reaction (hereafter PCR) has been performed indisposable reaction tubes such as small, plastic microcentrifuge tubesor test tubes which are placed in an instrument containing a thermallycontrolled heat exchanger. Examples of these instruments are disclosedin U.S. Pat. No. 5,038,852, U.S. application Ser. No. 07/709,374, filedJun. 3, 1991, and U.S. application Ser. No. 07/871,264, filed Apr. 20,1992, all of which are hereby incorporated by reference in theirentirety.

The heat exchanger in these instruments is typically a metal block;however, hot air ovens and water baths also have been used. Thetemperature of the reaction mixture in the reaction tubes is changed ina cyclical fashion to cause denaturation, annealing and extensionreactions to occur in the mixture. Three separate incubationtemperatures commonly were used in the first generation PCR thermalcycling applications. These were typically around 94° C. fordenaturation, around 55° C. for annealing, and around 72° C. forextension. More recently, the annealing and extension incubations havefrequently been combined to yield a two temperature incubation process,typically around 94° C. for denaturation, and around 50°-65° C. for anannealing and extension incubation. The optimal incubation temperaturesand times differ, however, with different targets.

Rapid, small scale, PCR capillary tube instruments also have appeared.For example, Idaho Technology introduced an instrument wherein thereaction mixture is placed in capillary tubes which are then sealed andplaced in a hot air oven which cycles the temperature of the reactionmixtures in the tubes. A similar system was described in a paper byWittwer et al., "Minimizing the Time Required for DNA Amplification byEfficient Heat Transfer to Small Samples", Analytical Biochemistry 186,328-331 (1990). There, 100 microliter samples placed in thin capillarytubes were placed in an oven with a heating coil, a solenoid activateddoor and a fan. Air was used as the heat transfer medium. A very similarsystem was also described by Wittwer et al in another paper entitled"Automated Polymerase Chain Reaction in Capillary Tubes with Hot Air,"Nucleic Acids Research, Volume 17, Number 11, pp. 4353-57 (1989).

The PCR volume has been limited to a range of from about 10 microlitersto 1.5 milliliters in conventional heat block or liquid bath heatexchanger PCR instrument designs where the reaction mixture has beenstored in microcentrifuge tubes. It is hard to scale up these volumes.The difficulty resides in the fixed dimensions of the wells in the heatexchange block for the tubes and the escalating difficulty in achievingheat transfer uniformity among all wells as dimensions get larger andheat gradient problems become more pronounced. As the volume of priorart reaction vessels is increased, the surface/volume ratio decreases.This change reduces the ability to change quickly the temperature of thereaction mixture in each tube because most heat exchange occurs betweenthe walls of the tubes and the walls of the wells in the sample block.

In prior art instruments, thermal ramps were long because there wassubstantial lag in the temperature of the sample relative to the blockcaused by poor convection and conduction. Substantial thermal rampdurations between incubation temperatures were often necessary toprevent significant temperature gradients from developing because of thelarge thermal mass of the metal blocks used in many instruments as wellas nondiffuse heat sources and sinks. These temperature gradients cancause non-uniform amplifications in different samples located at diversepoints along the temperature gradient. There is no chemical orbiological reason for using temperature ramps.

A capillary tube PCR instrument has the advantage of rapid thermalincubation transitions because the reaction volume and samplecontainment thicknesses can be minimized. One such instrument isdisclosed in U.S. Pat. No. 5,176,203, issued to D. M. Larzul. The Larzulpatent discloses a wheel shaped apparatus for automatic thermal cyclingof a fluid sample contained in a closed loop or spiral coil ofcontinuous capillary tube. Each loop of the tube is routed through threethermostatted zones. The sample is pushed through the loops by amotorized magnetic system in which a magnet on the end of a rotatingcentral arm magnetically pulls a slug of mineral oil containingsuspended metallic particles through the capillary tube. Since the slugabuts the sample in the capillary tube, the slug pushes the samplethrough the loop. The motorized system may be micro-processor controlledto regulate the movement of the sample in accordance with apredetermined protocol.

SUMMARY OF THE INVENTION

A number of alternative embodiments of capillary tube PCR instrumentsare envisioned herein which share certain common advantages. All of theinstruments disclosed herein use thin walled capillary tubes to hold thereaction mixture as opposed to microcentrifuge tubes. A capillary tubeas used herein is a tube which has an internal diameter less than 3 mmand preferably on the order of about 1 mm to 2 mm in internal diameter.These capillary tubes are heated and cooled in the embodiments taughtherein according to a user-defined PCR protocol required of a particularreaction mixture fed into a programmable computer which, in turn,automatically controls sample handling, flow, velocity, pressure, andtemperature to implement the protocol via conventional controlprogramming. The differences among the various embodiments of theinvention arise out of the different means used to heat and cool the PCRreaction mixture and different tube and fluid handling means used tomove the reaction mixture.

For example, a first embodiment of the invention automatically pumps thereaction mixture repetitively through a continuous loop of capillarytube which is routed through two different thermostatted fluid baths,one at a denaturation temperature and one at an anneal/extendtemperature. Additional baths could be added to increase the number ofincubation temperatures.

A second embodiment directs the reaction mixture through a capillarytube only once, i.e. in a single pass. The capillary tube is routed inan alternating fashion back and forth between a first thermostatted heatexchanger held at a denaturation temperature and a second heat exchangerheld at an anneal/extend temperature. Alternatively, a third heatexchanger may also be used if the anneal and extend temperatures differ.A positive displacement or peristaltic pump or syringe is used to pushthe reaction mixture through the single pass tube out into a productcollection vessel.

A third embodiment of the invention involves a stationary reactionmixture in a multiple fluid bath arrangement. This embodiment uses asingle loop of capillary tube for each sample. The portions of the loopscontaining the samples are enclosed in a reaction chamber. A hot fluidat a denaturation temperature is pumped into this reaction chamber froma first thermostatted fluid bath and held there during the denaturationincubation. Fluid at an anneal/extend temperature is pumped into thechamber from a second temperature stable bath after removal of the hotfluid from the first temperature stable bath to implement theanneal/extend incubation. This process is repeated as necessary tocomplete the PCR protocol.

A fourth embodiment utilizes two or three temperature stable fluidbaths, each of which is constantly circulating its fluid through aseparate conduit. Each fluid stream is thermostatted at one of thenecessary PCR incubation temperatures. A single cylindrical heatexchanger chamber with a wire mesh at the input end is connected througha valve system to the fluid streams. The wire mesh holds individualsmall capillary tube reaction mixture vessels, which are each sealed atboth ends in place in the heat exchange chamber. The vessels are held ina spaced relationship or array by the mesh because the seal at one endof each capillary tube vessel is too large to fit through the mesh. Thevalve system, preferably under the control of a computer or otherautomated controller, is used to select one of the streams at a time tobe routed through the cylindrical heat exchanger chamber while the otherone or two streams are bypassed around it. For example, to carry out adenaturation incubation, a stream of 94° C. fluid is routed through theheat exchanger chamber while an anneal stream at 55° C. and an extendstream at 75° C. are routed around the heat exchange chamber.

A fifth embodiment uses two metal blocks each of which has itstemperature stabilized at one of two temperatures needed for thedenaturation and anneal/extend incubations. An open-ended, thin-walledcapillary tube is routed through and between these two metal blocks. Aperistaltic pump or a plunger and seal arrangement, similar to asyringe, is connected to one end of the capillary tube, under control ofa computer programmed to carry out the PCR protocol. The pump or plungeris activated back and forth to draw reaction mixture into the capillarytube, move it into the region surrounded by the denaturation block,i.e., the block held at the denaturation temperature, and then move thereaction mixture through the capillary tube into the region of thecapillary tube surrounded by the block held at the anneal/extendtemperature. This cycle is repeated the required number of times tocomplete the PCR protocol. The plunger is then moved to discharge thePCR product. Alternatively, the blocks themselves could be translatedback and forth against a stationary capillary tube containing themixture to thermally cycle the mixture.

A sixth embodiment of the capillary PCR instrument in accordance withthe invention is somewhat similar to the fifth in that spaced metal heatexchanger blocks are used. This embodiment preferably has a pair ofspaced heat exchanger blocks, a plurality of open-ended capillary tubesrouted through each of the blocks, and an automated sample handlingsystem. This handling system simultaneously inserts the end of each ofthe capillary tubes into reaction mixture containers, withdraws thereaction mixture into the capillary tubes, then translates the mixturebetween the heat exchanger blocks, and discharges the final PCR productfrom the capillary tubes into suitable containers, all under computercontrol. In addition, the handling system performs capillary tubecleansing, rinsing, and sample tray translation and elevation functions,all under computer control so that a tray of 48 or 96 samples can bethermally cycled automatically in groups, if necessary, e.g. 12 at atime.

A seventh embodiment of the invention is designed to move the heatsource rather than the sample. This embodiment has a moving thermal heatexchanger and preferably has a cylindrical heat exchange thermal platenor drum, divided into two, three, four, or more axially extending radialsegments. Each segment is thermostatically controlled at an appropriateincubation temperature for denaturation, extension, or annealing. Ifonly two incubation temperatures are required and three segments areprovided, the third segment may be used to accelerate the transitionfrom the anneal/extension temperature to the denaturation temperature.Similarly, if four segments are provided, and two incubationtemperatures are required, then the other two segments may be used toaccelerate the transitions in both directions between theanneal/extension temperature and the denaturation temperature.

The arcuate outer surface of each of the segments has a plurality ofparallel grooves, each aligned to receive a capillary tube. Thecapillary tubes are routed so that there is preferably full tube contactalong the groove length of one segment. A handling system is alsoincluded as in the sixth embodiment. A peristaltic pump or syringe typeplunger and seal assembly is used to draw the reaction mixture into eachof the capillary tubes to a position adjacent to the contactingcylindrical heat exchange segment. The PCR protocol is then performed byrotating the cylindrical heat-exchange platen between alternate angularpositions in which each segment is preferably in full contact with thecapillary tubes for the required incubation period.

All of the instruments described herein are preferably controlled bycomputers which are user-programmable, such as a personal computer (PC)or an internal microprocessor based controller of conventional design,adapted to receive data defining the desired PCR protocol and to carryout the protocol by issuing the proper control signals to operate thenecessary pumps and valve switches, and/or generate the heat exchangermedium movements needed to cause the reaction mixture to be heated andcooled.

All of the instruments described herein enjoy the advantage of notneeding to change the temperature of a large thermal mass in order tocycle the reaction mixture between the anneal/extend incubationtemperature(s) and the denaturation temperature. This simplificationreduces thermal gradient problems and simplifies the thermal design ofthe instruments such that plural reaction mixtures can all be subjectedto the same PCR incubations at the same time without variance caused bynonuniformities in the instrument itself.

The primary portions of the instruments of the present invention whichundergo substantive thermal transients are the capillary tube walls.Because of the low thermal mass and high thermal conductivity of theseportions of the instruments, very rapid thermal cycling of samples canbe carried out. Use of capillary tubes in most of the instrumentsdescribed herein also permits simple scaling of the amount of finishedproduct without the need for heat exchanger redesign to minimizenonuniformities in temperature among plural samples being processed.

The capillary tube PCR instruments disclosed herein enjoy the advantagegenerally of being capable of very rapid temperature changes for thereasons described above. This improvement shortens the overall time fora PCR cycle and considerably shortens the overall time for a completeamplification protocol. These and other objects, features, andadvantages of the invention will become more apparent from a reading ofthe following detailed description when taken in conjunction with thedrawing and appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a block diagram of a capillary tube PCR thermal cyclerinstrument in accordance with a first embodiment of the presentinvention.

FIG. 2 is a block diagram of a capillary tube PCR instrument inaccordance with a second embodiment of the present invention.

FIG. 3 is a block diagram of a capillary tube PCR instrument inaccordance with a third embodiment of the invention for performing smallscale, very rapid PCR.

FIG. 4 is a block diagram of a capillary tube instrument in accordancewith an alternative third embodiment of the invention.

FIG. 5 is a block diagram of a capillary tube PCR apparatus inaccordance with a fourth embodiment of the invention using threethermostatted circulating fluid baths and a single reaction chamber.

FIG. 6 is a simplified sectional view of the reaction chamber of theapparatus shown in FIG. 5 taken along the line 6--6 with all flowsbypassed.

FIG. 7 is a simplified sectional of the reaction chamber of theapparatus as in FIG. 6 with the 55° C. flow directed through thereaction chamber and all other flows bypassed.

FIG. 8 is a simplified sectional of the reaction chamber of theapparatus as in FIG. 6 with the 75° C. flow directed through thereaction chamber and all other flows bypassed.

FIG. 9 is a simplified sectional of the reaction chamber of theapparatus shown in FIG. 6 with the 95° C. flow directed through thereaction chamber and all other flows bypassed.

FIG. 10 is a simplified view of the reaction chamber of the apparatusshown in FIG. 5 showing an exemplary single capillary reaction tube heldin place by a wire mesh.

FIG. 11 is a diagram of an apparatus for injection of a reaction mixtureinto a capillary tube in graduated amounts.

FIG. 12 is a conceptual diagram of a fifth embodiment of a capillarytube PCR instrument using two metal block heat exchangers held atconstant temperature and a capillary tube reaction chamber runningtherebetween in which the reaction mixture is pumped back and forth.

FIG. 13 is a diagram of the PCR instrument shown in FIG. 12 at thedenaturation stage of a PCR protocol.

FIG. 14 is a diagram of the PCR instrument shown in FIG. 12 at theanneal/extend stage of a PCR protocol.

FIG. 15 is a diagram of the PCR instrument shown in FIG. 12 at thefinished product ejection stage of a PCR protocol.

FIG. 16 is a diagram of an alternative fifth embodiment of the PCRinstrument shown in FIG. 12 at the sample introduction stage of a PCRprotocol.

FIG. 17 is a diagram of the PCR instrument shown in FIG. 16 at thedenaturation stage of a PCR protocol.

FIG. 18 is a diagram of the PCR instrument shown in FIG. 16 at theanneal/extend stage of a PCR protocol.

FIG. 19 is a diagram of the PCR instrument shown in FIG. 16 at thesample ejection stage of a PCR protocol.

FIG. 20 is a simplified perspective view of a sixth embodiment of a PCRapparatus for an array of capillary reaction tubes in accordance withthe invention.

FIG. 21 is an enlarged partial side view of the sample translationassembly in the apparatus shown in FIG. 20.

FIG. 22 is an enlarged side view of the sample transfer device in theapparatus shown in FIG. 20.

FIG. 23 is a simplified side view of a seventh embodiment of a capillaryPCR apparatus having a rotating drum thermal heat exchanger for an arrayof capillary reaction tubes in accordance with the invention.

FIG. 24 is a perspective view of a breadboard version of the capillaryPCR apparatus shown in FIG. 23.

FIG. 25 is an enlarged partial front view of the rotating drum thermalheat exchanger assembly in the apparatus shown in FIG. 24.

FIG. 26 is a partial side view of the rotating thermal heat exchangerassembly shown in FIG. 24.

FIG. 27 is an enlarged partial rear view of the heat exchanger assemblyshown in FIG. 24 in accordance with the invention.

FIG. 28 is a partial sectional view of the sample handling assemblyshown in FIGS. 20 and 24.

FIG. 29 is an enlarged sectional view of the tip of a capillary tubeshown in FIG. 28.

FIG. 30 is a schematic view of a detection system coupled to the seventhembodiment of the invention.

FIG. 31 is a flow diagram for the control software for the seventhembodiment of the present invention.

FIG. 32 is a flow diagram of the sample load subroutine portion of thediagram shown in FIG. 31.

FIG. 33 is a flow diagram of the thermal cycling subroutine referred toin FIG. 31.

FIG. 34 is a flow diagram of the sample unload subroutine referred to inFIG. 31.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Continuous LoopCapillary Thermal Cycler

Referring to FIG. 1, there is shown a first embodiment of acapillary-tube PCR instrument in accordance with the invention. Theinstrument 10 comprises two different thermostable liquid baths with thereaction mixture pumped therebetween in a capillary-tube loop. Thisembodiment is capable of performing PCR on a reaction volume of anyscale including greater than about 1 milliliter. Two reactiontemperatures are used for the denaturation and annealing/extensionincubations. Complete control over the incubation times at eachtemperature is provided by a pump 12 and the ratio of tube length ineach bath.

Typical two-temperature PCR involves cycling a reaction mixture betweenabout 60° C. and 95° C. with residence or incubation times in the rangeof 1-300 seconds. Three temperature stable liquid baths may be used forthree separate incubation temperatures. Kim and Smithies published anarticle in Nucleic Acids Research 16, 8887-8903 which teaches thatincubation at the intermediate "extension" temperature is unnecessary.Accordingly, the embodiment of FIG. 1 uses only two temperatures basedupon this research, one in the range from 37°-72° C. for annealing andextension and one in the 85°-98° C. range for denaturation.

The first embodiment is designed for preparative scale PCR with volumesupward of about 1 milliliter with no set upper bound other thanpotentially the maximum volume of the reaction tube loop. The preferredtarget DNA segment may have between about 100-10,000 base pairs (bp).The first embodiment is designed for optimized consumption of thestoichiometrically limiting and most expensive reagents needed for thePCR reaction mixture, i.e., the two primers.

In FIG. 1, the high temperature bath is shown at 16 and the lowtemperature bath is shown at 18. These two baths are separated by alayer of insulation 20 which is selected to minimize the flow of heatbetween the two baths 16 and 18. Bath 16 is thermostatically controlledto a user-defined temperature preferably between 85° and 98°C.±0.1°-0.5° C. Bath 18 is thermostatically controlled at a user-definedtemperature between 40° C. and 70° C., preferably within the sametolerance. Each bath may employ turbulent mixing via conventional meanssuch as mixer blades, etc. to achieve thermal uniformity.

A reaction vessel 22 comprises a length of relatively thin-walledplastic capillary tubing which forms a loop which penetrates the wallsbetween the two baths through two O-ring seals 24 and 26, submergedbelow the liquid levels. The inner diameter of the reaction tube 22 andits length establish the theoretical upper boundary for the reactionvolume. Narrower tubing of greater length is preferred to achieve bettertemperature control.

A 4-way valve 14 is immersed in the liquid within the low temperaturebath 18 and is used to alter the fluid flow path to introduce a reactionmixture sample into the reaction vessel 22 and remove finished PCRproduct. A peristaltic pump 12 also located in bath 18 is used tocirculate the reaction mixture into, out of, and through the reactionvessel 22. The pump 12 has a closely controlled and calibrated flowrate. Peristaltic pumps are preferred so that the reaction mixturecontacts only plastic.

If the tubing used in both baths has the same internal and externaldiameters, the residence time in each bath for the reaction mixture iscontrolled by the ratio of the tubing lengths in the two baths.Generally, it would be preferable to use denaturation incubation timesin bath 16 of 5-15 seconds and annealing/extension incubation times ofup to several minutes in bath 18. In this first embodiment, a singlecontinuous reaction tube 22 passes between the two baths through leakproof O-ring seals 24, 26 and both ends connect to the 4-way valve 14.The use of O-ring seals 24, 26 through which the reaction tube can slideallows incubation times to be adjusted manually by pulling the tubethrough the O-rings to adjust the relative lengths of tube in each bath.

The 4-way valve has a position in which reaction product from the lengthof tube 22 in the low temperature bath 18 can be pumped back into thehigh temperature bath 16 to commence a new cycle, a position fordischarge of PCR products at the completion of a PCR protocol, and aposition where new reaction mixture can be introduced to the reactiontube 22 after suitable procedures are performed to flush, clean or, ifnecessary, replace the reaction tube 22.

An immersible, fiber-optic spectrophotometric detector 28 may beincluded, preferably having its sensor in the low temperature bath 18,to monitor the progress of the PCR reaction from one cycle to another.This spectrophotometric (UV) detector 28 detects the hypochromismexpected as dNTPs are incorporated into the DNA product. Only a marginaldifferential signal will typically be seen until the later thermalcycles in the PCR protocol. A similar fluorescence detector may also beused in performance of various "Tagman" based assays.

Reaction tube 22 should be a loop of capillary tubing made ofPCR-compatible material such as Polytetrafluoroethylene (PTFE), e.g.DuPont Teflon®, which has an inside diameter of less than 3 mm and ispreferably between about 1 mm and 2 mm. The tubing should have a wallthickness between 0.2-0.5 mm and is preferably about 0.3 mm. Thecapillary size of the tube 22 minimizes the effects of the heat transfergradient across the tube wall as the reaction mix passes through thebath. In addition, the small size permits the use of an air bubble or animmiscible fluid such as an oil to be used as a discontinuity to demarkthe ends of the reaction mixture sample volume and as a pusher to drivethe reaction mix predictably through the baths 16 and 18 via theperistaltic pump 12 and through the 4-way valve 14.

The bubble or other fluid discontinuity travelling through the reactiontube 22 can be used to provide a simple, automatable cycle counterthrough use of suitable apparatus to detect the passage of the fluiddiscontinuity. Suitable apparatus to detect the passage of thediscontinuity would include a photoelectric or capacitance sensor 42mounted on the tube 22, again, in both.

Operation of the first embodiment is simple. After the reaction mixtureis introduced through the 4-way valve 14 into the tube 22, it iscontinuously pumped through the thermostatted baths 16 and 18 with atotal cycle time defined by the speed of the pump 12 and the tubingvolume. Each passage of the mixture through the complete loop is onecomplete PCR cycle. The entire reaction volume may not be at a singletemperature at any one time, but each volume element of the reactionmixture experiences approximately a uniform residence time at eachtemperature. Because of the relatively narrow tubing bore and surfaceroughness, there will be some turbulent mixing within the mixturesegment as it passes through the tubing. This mixing enhances thesharpness of the thermal control. After the desired number of cycleshave been completed, the finished PCR product is pumped out through the4-way valve.

Commercially available thermostatted baths can regulate the bathtemperature within 0.1° C., thereby offering precise temperaturecontrol. No special flow controls or software are needed in thisembodiment since peristaltic pumps with very precise flow rates are alsocommercially available.

It may be desirable to automatically alter the annealing/extensionincubation temperature or time late in the PCR protocol after largeamounts of finished product have begun to build up in the reaction tube22. Automated or manual changes in the pump flow rate or the lower bathtemperature setpoint may be implemented through a suitably programmedCPU 30 coupled to a pump speed control input and a bath temperaturecontrol input through a suitable interface. It may also be desirable togradually increase the denaturation temperature over the completeduration of an amplification, in a manner that is linear with totalelapsed time. This protocol may be accomplished through a suitablyprogrammed CPU coupled to a temperature control input of the hightemperature bath 16 and to a cycle counter. Such a control scheme issymbolized schematically by the CPU 30 shown in FIG. 1 in dashed lineswith control lines 32, 34, and 36 coupled to the low temperature bath18, the peristaltic pump 12, and the high temperature bath 16,respectively.

It may also be desirable to accommodate supplemental reagent additionssuch as dNTPs, enzyme or primers throughout the PCR protocol or in thelater cycles in some alternative embodiments. This may be doneautomatically under the control of CPU 30 via a reagent addition valvingmechanism symbolized schematically by block 38 controlled via signals oncontrol bus 40. Various embodiments of the valving mechanism 38 mayprovide for continuous addition of reagents or addition only in latercycles. The CPU 30 is coupled to a cycle counter 42 via a control bus 44in embodiments where the number of cycles completed needs to bemonitored, such as embodiments where reagents are to be added atspecific points later in the PCR protocol. Reagent additions later inthe protocol can maximize the total yield. For example, fidelity can beimproved by keeping the dNTP concentrations in the 10-50 micromolarrange, but these levels might prove to be stoichiometrically limiting inlater cycles. Therefore, an order-of-magnitude jump in Mg-dNTPconcentration late in the amplification protocol may improve yielddramatically with little net effect on fidelity. Similarly, primerconcentration may be increased in late cycles to boost total yield. Thisembellishment would improve product purity as well.

The inside surface of the capillary tubing creates drag on the portionof the sample liquid adjacent the tube wall. An air bubble or otherfluid discontinuity is useful between each sample to push the entiresample as a unit along the capillary flow path. Otherwise a parabolicflow profile would exist across the capillary. Use of an air bubblebetween samples or between sample segments results in "slug" flowwherein sample "slugs" are pushed through the capillary tubes.

Single-Pass Capillary Thermal Cycler

A simplified schematic diagram of a second embodiment of a capillarytube PCR instrument for performing PCR in comparatively large volumes isshown in FIG. 2. This embodiment uses a length of capillary tubing 50that is routed in and out of hot and cold sources of the appropriatetemperature a number of times. The embodiment of FIG. 2 uses a reactionchamber in the form of series loops of capillary tubing 50. Each loophas a portion which passes through an equilibrated hot zone 52 and aportion which passes through an equilibrated cool zone 54. Typically,the hot zone 52 is a liquid bath wherein the liquid is controlled tohave a constant temperature in the range of temperatures needed toperform denaturation in PCR processes, typically around 95° C. Likewise,the cool zone is usually a liquid bath with liquid therein having itstemperature controlled so as to maintain a constant temperature in therange of temperatures needed for annealing and extension, typicallyaround 60° C.

Other temperatures could be used within the denaturation andanneal/extend ranges, and each of the hot and cool zones 52,54 could betemperature controlled solid blocks through which the tubing 50 passes,such as a metal block. Also, a hot gas oven could be used fordenaturation and a cool gas oven used for the cool zone. The preferredstructure for the hot and cool temperature controlled zones, however, iseither two temperature-controlled metal blocks or twotemperature-controlled water baths. The length of the tubing 50, itsinternal diameter, the length of tubing in each of the hot and coolzones, and the flow rate of reaction mixture inside the tube are allfactors in the control of the amplification.

In the second embodiment of FIG. 2, the motive force for moving the PCRreaction mixture along the tubing 50 is a sterile, disposable syringe 51capable of holding large volumes of reaction mixture 53. The syringe 51is loaded with the reaction mixture components well known to thoseskilled in the art, and the sample nucleic acid to be amplified. Thesyringe 51 is then operated by hand, compressed gas, or using a syringepump to move the reaction mixture 53 into and through the tubing 50.

Another syringe 55 containing air or an immiscible fluid such as mineraloil may be connected in parallel with the syringe 55 to the tubing 50.The syringe 55 is operated intermittently to inject air bubbles orimmiscible fluid bubbles into the flow stream of reagent mixture 53 inthe tubing 50. This will ensure that slug flow dominates throughout theflow path so that each volume element of the reaction mixture 53experiences the same number and duration of thermal incubations. The useof air to cause slug flow to occur is preferred as, upon fluid exit,only the finished PCR product is collected in the container 56. The useof the syringe 55 may not be required for preparative-scale PCR; as longas enough cycles are run to exhaust the concentration of primers and aslong as the dynamic residence time within each zone is sufficient tocause most of the sample to reach incubation temperature. The syringe 55may also be used to introduce a missing reagent to effect a "hot start".

The length of the tubing 50 in each zone is chosen so that each time aunit of volume of the reaction mixture has passed once through the hotzone and once through the cool zone, one complete PCR cycle will havebeen completed. The number of loops in the system determines the numberof cycles performed. The preferred method of operation includes leavingan air pocket at the top of the syringe 51 to force all the amplifiedproduct out of and completely through the tubing.

The tubing of the reaction tube 50 may be either glass (coated with anappropriate silanizing agent), metal, or plastic of a type which willnot interfere with the PCR process. Preferably, the tubing is of thesingle-use, disposable variety. Plastic, polypropylene or teflon ispreferred. However, metal tubing, coated with parylene, may be preferredin this embodiment because it has better heat conductance, it holds itsshape after being bent, the internal diameter of the tubing can besmaller so as to provide more turbulent mixing during slug flow, andmetal is more thermally compatible with a heated block than is plastictubing.

The principal advantage of the second embodiment shown in FIG. 2 is thelarge volume of amplified product that can be obtained in one pass. Itwould be more convenient to operate than the conventional type of PCRinstrument wherein many individual 100 microliter reaction mixes must beprepared to obtain a large volume of amplified product. The individualreaction mixtures conventionally must be prepared, amplified, pooled,and realiquoted, which is a tedious and time-consuming procedure.

Multiple Water Bath Capillary Thermal Cycler

A diagram of a third embodiment of a capillary tube PCR instrument inaccordance with the invention for performing relatively small-scale,very rapid PCR using water baths to change the temperature of a chamberin which the capillary tube resides is shown in FIG. 3. In thisinstrument, a liquid tight chamber 60 contains at least one capillarytube reaction chamber 62 which has both ends 64 and 66 protruding fromthe chamber through liquid-tight grommets. As is the case for all thecapillary tube instruments disclosed herein, the capillary tube 62 maybe made of metal, preferably parylene coated, or plastic, with Teflon orpolypropylene preferred for the plastic embodiments. From 1 to 25microliters of the PCR reaction mixture is injected into the portion ofthe capillary tube 62 in the chamber 60 by any suitable means.

The chamber 60 is in fluid communication with either two or threetemperature controlled fluid baths. In FIG. 3, only two baths are shown:a high-temperature bath 68 kept at a constant temperature in thedenaturation temperature range such as 95° C.; and a cooler bath 70 keptat a constant temperature in the annealing/extension range such as 60°C. Embodiments where three separate incubations for denaturation,annealing, and extension processes are desired require a thirdtemperature-controlled fluid bath 72 indicated by the dashed lines inFIG. 3.

Each fluid bath may have its own integral fluid pump coupled to inputand output pipes such as pipes 74 and 76, respectively, for bath 68. Inthese embodiments, suitable control circuitry (not shown) is used topump hot fluid from bath 68 into chamber 60 to a sufficient level tofully immerse the capillary tube 62 for a predetermined period of timefor the denaturation process to occur, typically from 1 second to 3minutes. Water flow into and out of the chamber is typically set at atleast one liter per minute to make switches between temperatures faster.

After completion of the denaturation incubation, suitable controlsignals are issued to cause the 95° C. fluid in the chamber 60 to bepumped back into the hot fluid bath 68, and, when the level of fluid inthe chamber 60 is sufficiently low, suitable control signals are issuedto cause the cooler fluid from the bath 70 to be pumped into the chamber60. A liquid level sensor mechanism such as a float switch (not shown)can be used to determine when to switch on the pump in the reservoir 70to begin filling the chamber 60 with fluid of a temperature to carry outan anneal/extend incubation. The reaction mixture changes temperaturerapidly when the chamber 60 is filled with a different-temperature fluidbecause capillary tube is used which constitutes a small thermal mass.In this third embodiment, the fluid from each water bath 68, 70, or 72may be pumped back into its original bath 68, 70, 72 when the incubationat that temperature is over. However, this procedure leaves thecapillary tube 62 temporarily suspended in air before the fluid from thenext bath enters the chamber 60. The relative volumes of the chamber 60and the water baths are such that the volume of the chamber 60 is tinycompared to the volume of the water baths. Therefore it is preferable toroute the fluid from the chamber 60 to the water bath which is providingthe new fluid for the next incubation without prior drainage of chamber60. In other words, fluid may be simply transferred through the linesbetween each bath and the chamber for each incubation. Even though thetemperature of the fluid from the chamber 60 is different from thetemperature of the fluid in the fluid bath being pumped in for the newincubation, the thermal mass of the water baths is so large that thereis no significant change in the bath temperature, and the bath controlscan maintain constant bath temperatures. This design permits temperaturechanges to be accomplished typically in less than 1 second.

FIG. 4 represents an alternative third embodiment of a capillary-tubePCR instrument similar to the third embodiment shown in FIG. 3, using asingle pump to pump the fluid from any of the baths. Like-numberedelements in FIGS. 3 and 4 mean that the structures are the same and thatthey perform the same function. In FIG. 4, a single bi-directional pump78 is coupled to the chamber 60 by input pipe 80 and output pipe 82.This pump is also coupled to the output port of a fluid multiplexer 84by input pipe 86 and output pipe 88. The fluid multiplexer 84 has aplurality of input ports, each of which is coupled to one of the two orthree temperature-controlled baths 68, 70, and 72. Each input port iscoupled to its corresponding fluid bath by two pipes such as input pipe86 and output pipe 88 for fluid bath 68.

The fluid multiplexer 84 and pump 78 are controlled by a suitableconventional programmable controller (not shown) with suitable interfacecircuitry to carry out the appropriate position selection of one of thefluid flow input ports for coupling to the fluid flow output port andappropriate switching of direction of pumping by the pump 78 so as tocarry out the desired PCR protocol of sequential incubations.

The instruments shown in FIGS. 3 and 4 can perform PCR amplification onvolumes under 10 microliters without using an oil or other fluiddiscontinuity means because the mixture is stationary during the PCRprotocol. The instruments shown in FIGS. 3 and 4 can also perform PCRincubation steps in as little time as 1 second, and total time for atwo-incubation PCR protocol cycle can be reduced to as little as 8seconds by the use of 1 second denaturation and anneal/extend times offrom 7-30 seconds. When anneal/extend intervals are this short, however,large concentrations of thermostable enzyme such as Taq polymerase maybe required in the sample volume.

Constant Circulating Fluid Bath Capillary Thermal Cycler

A fourth embodiment of the capillary tube PCR instrument of theinvention is shown in FIG. 5. This illustrated embodiment has threethermostatted fluid baths and a single reaction chamber which containssealed capillary tubes holding the reaction mixture samples. If only twotemperatures are needed for the PCR protocol, however, the third bathcan be eliminated. The third bath is described herein for completeness.

A first bath 98 maintains a body of fluid at some temperature in therange of annealing temperatures such as 55° C. A second bath 100maintains the temperature of fluid contained therein at a temperature inthe range of temperatures effective for the extension incubation, forexample 75° C. A third bath 102 maintains the temperature of fluidcontained therein at a temperature in the range of temperatureseffective for denaturation, typically 95° C. Each fluid bath has a pumptherein which can pump the fluid in the bath out an output pipe 108,through a valve mechanism 104 at the input side of a reaction chamber106. Similarly, the output pipe 110 of bath 100 and the output pipe 112of bath 102 connects to valve mechanism 104. Fluid from reaction chamber106 returns to its bath through an output valve mechanism 120 and eachbath includes a return pipe, i.e., pipes 114, 116, and 118 for baths 98,100, and 102, respectively.

A computer 122 is coupled via a control bus 124 to the pumps of thevarious baths to control the fluid flow rate in the preferredembodiment. Constant flow rates may also be used in alternativeembodiments, in which case coupling of the pumps to the computer 122 maynot be necessary as the pumps will be running continuously at the sameflow rate.

The computer 122 is also coupled to the input valve mechanism 104 andthe output valve mechanism 120. The purpose of the computer control ofthe valve mechanisms is to selectively direct the flow of onetemperature fluid into the reaction chamber 106 and bypass the otherflows in accordance with data stored in the computer 122 defining thedesired PCR protocol.

As with other PCR instruments such as is described in U.S. Ser. No.07/871,264, filed Apr. 20, 1992, the PCR protocol may be defined bycheckpoint data defining the temperature and duration of all therequired PCR incubations, the number of cycles to perform, and linkingdata to any other desired protocols to run upon completion of any givenprotocol. The data defining the desired protocol may be entered by theuser or it may be a predefined program.

The PCR protocol is implemented by the computer 122 by controlling theinput valve mechanism 104 such that only one flow from one of the bathsis directed through the reaction chamber at any particular time, and allthe other flows are bypassed through alternate pathways that do not passthrough the reaction chamber 106. Valve mechanism 104 may be acylindrical or rotary solenoid-operated or air-operated multiport valvewell known to those skilled in the art. The flow sequence is betterunderstood by reference to FIGS. 6-9 which shows one arrangement of thereaction chamber 106 and the alternate flow paths in various states ofbypass.

The tubular reaction chamber 106 preferably has the bypass piping fromthe two or three baths 98, 100, and 102, also tubular, mounted aroundthe outside of the chamber 106. Valve mechanisms 104 and 120 are atopposite ends of chamber 106. A transverse sectional view through thisarrangement would show the interior of chamber 106 surrounded by thebypass piping. This sectional view is schematically illustrated in FIGS.6 through 9.

It is to be understood that if the anneal and extension temperatures arethe same, then only two baths and bypass paths would be necessary andthe instrument in FIGS. 5 through 9 would required only two rather thanthree bypass paths.

FIG. 6 shows the reaction chamber 106 empty, with all three flowsbypassed through their alternate pathways. The anneal temperature fluidflows through bypass pathway 130, the extension temperature fluid flowsthrough bypass pathway 132, and the denaturation temperature fluid flowsthrough the bypass pathway 134.

FIG. 7 reflects the valve configuration for the anneal incubation. Inthis incubation, the 55° C. fluid from bath 98 is switched via computer122 by solenoid-operated valves in valving mechanism 104 so as to passthrough the reaction chamber 106. The temperature of the reactionmixtures in the capillary tubes in the reaction chamber is then rapidlyequilibrated at 55° C. Both the 75° C. fluid from bath 100 and the 95°C. fluid from bath 102 remain connected to their bypass pathways 132 and134, respectively.

After the appropriate annealing incubation interval has passed, thecomputer 122 activates the valve mechanisms 104 and 120 to switch theflows to the configuration shown in FIG. 8 to carry out the extensionincubation. In this incubation, the 95° C. and 55° C. fluid flows arebypassed and the 75° C. flow is switched to pass through the reactionchamber 106.

Upon completion of the extension incubation, denaturation to split thedouble-stranded extension product into single stranded templates for thenext cycle must be performed. The computer 122 again activates valves104 and 120 to switch the flows to the configuration shown in FIG. 9.Here the 55° C. fluid flow and the 75° C. fluid flow are bypassed, andthe 95° C. fluid flow is switched to pass through the reaction chamber106. This step completes one PCR cycle. The computer 122 then typicallyrepeats the cycle the desired number of times to complete the PCRprotocol.

The reaction chamber 106 is a tubular chamber with a wire screen meshtransversely mounted in the reaction chamber at the input end. Thereaction mixture is stored in thin-walled (0.01-0.03 mm) sections ofseamless, inert, puncture-free plastic capillary tubing of variousdiameters depending upon the desired volume of finished product. Insidediameters up to 2 mm and as small as 0.075 mm are available. The tubingtypically has a maximum continuous service temperature rating of 300° C.and is not affected by body fluids. For example, tubing which may beused for this purpose is commercially available from Micro ML TubingSales, 45-10 94th Street, Elmhurst, N.Y. 11373.

The reaction mixture is injected into each of the tubes and each tube isclosed at both ends, preferably with a bubble at each end so as toisolate the reaction mixture from the ends of the tube. The clamp orother closure at one end is small enough to fit through the wire meshscreen. The clamp at the other end is preferably too large to fitthrough the screen. After injecting the reaction mixture into as many ofthe tubes as desired, a plurality of the capillary tubes are threadedthrough and suspended by the wire mesh. The bundle of tubes and the meshare installed in the reaction chamber 106 and the chamber closed andsealed. The clamp 144 is simply a heat sealed end of the capillary tube140 which forms an enlarged clamp. The computer is then activated tobegin switching the fluid flows through the chamber 106 around thecapillary tubes as above described.

FIG. 10 shows an exemplary capillary tube arrangement in the reactionchamber 106. In this Figure, a single capillary tube 140 is shown heldin place in the reaction chamber 106 by a wire screen 142 across one endof the reaction chamber 106. The tubing clamp 144 at the upstream end ofthe capillary tube 140 holds the capillary tube 140 in place with thedownstream end free to wiggle in the turbulent flow within the reactionchamber 106 because it is too large to fit through the mesh of thescreen 142 as mentioned above. Bubbles 146 and 148 in the ends of thecapillary tube 140 isolate the reaction mixture 150 from the ends of thetube. In this preferred embodiment, the upstream clamp 144 also bears acoded tab or stamp which served to identify the sample. Thepaddle-shaped clamp 144 also flutters in the fluid flow, and thuscreates turbulence as the circulating fluid passes it.

FIG. 11 shows a system for loading the capillary tubes 140 with knownamounts of PCR reaction mixture and removing the PCR product afterprocessing. A syringe 151 is inserted into the mouth 152 of thecapillary tube 140 with an air-tight fit. The capillary tube 140 may begraduated itself or may be placed in a holder 154 with a graduated scalenext to the tube as shown. The open end of the capillary tube 140 isimmersed in a reservoir of the reaction mixture 150. The syringe plungeris then withdrawn, sucking enough air out of the capillary tube to lowerthe pressure therein below atmospheric pressure. The PCR reactionmixture is then pushed into the capillary tube 140 by the higheratmospheric pressure outside acting on the reaction mixture reservoir.The level or amount of reaction mixture is determined by the position ofthe meniscus within the capillary tube 140. The position of the meniscuswill indicate volume uptake within ±2-5%. After filling the capillarytube 140, the ends are sealed, clamped, or capped and PCR processingproceeds. Removal of the finished PCR product is accomplished by cuttingthe end clamps off the capillary tube 140 and reversing the aboveprocess. Note that, in this process, the capillary tube 140 is its ownpipette. The syringe never touches the reaction liquid, therebyminimizing the chance of cross contamination.

The fourth embodiment described with reference to FIGS. 5 through 9 hasthe advantages of being able to process different sample volumes fromanalytical, to semi-prep scale, and to preparative scale, all at thesame time. The volume processed can be changed by altering the capillarytube length, the number of tubes in the reaction chamber, and/or theinternal diameter of the tubes. Also, turbulent flow in the reactionchamber accelerates reaction mixture temperature equilibration after atemperature step. Preferably the volume of the reaction chamber is lessthan 10% of each bath's volume. Variable control by the computer 122over the flow rate through the chamber can permit optimizing the timeresponse and temperature equilibration time between PCR runs acting ondifferent numbers of capillary tubes and sample volumes where differentpercentages of the reaction chamber volume are occupied by sample tubes.Very rapid and precisely controlled temperature changes in thetemperature of the reaction mixtures in the capillary tubes may beachieved because of the high precision of the fluid bath temperaturecontrol, for example, bath precision of ±0.1° C. is availablecommercially. In addition, the low heat capacity of the capillary tubes,the high rate of heat flow between the reaction mixtures and the heatexchanger fluid, the turbulent flow around the capillary tubes, and theabsence of voids in the chamber 106 all aid in minimizing the total PCRprotocol time.

An excellent advantage of the instrument shown in FIG. 5, as with manyother of the capillary tube PCR instruments disclosed above, is theability to scale up the volume of finished product produced without theneed to engineer a new thermal design for a metal block heat exchangerand without disturbing the thermal cycle parameters. This flexibilitycan ease the complications of using prior art PCR instruments forPCR-based manufacturing processes. The instrument design shown in FIG. 5is also inexpensive to build because substantially all of itscomponents, such as the water baths, personal computer, solenoid valves,capillary tubing, syringe, etc., are readily available, off-the-shelfitems.

Metal Block Capillary Thermal Cycler

A fifth embodiment of a capillary tube PCR thermal cycler instrument inaccordance with the invention is shown in FIG. 12. In its simplest form,this device comprises two metal block heat exchangers 170 and 172separated by a layer of insulation 174. The heat exchangers 170 and 172could also be other types of heat exchangers such as thermostaticallycontrolled constant temperature fluid baths. Each metal block heatexchanger 170 and 172 is preferably made of aluminum or some other goodheat conducting metal to minimize temperature gradients therein.

The temperature of each metal block is maintained at a constanttemperature by any suitable temperature control system such as issymbolized by box 176 for metal block 170 and box 178 for metal block172. The temperature of metal block heat exchanger 170 is maintainedconstant in the temperature range suitable for PCR denaturation,typically 92°-98° C. and typically 94° C. by temperature control system176. The temperature of metal block heat exchanger 172 is maintainedconstant somewhere in the range of temperatures suitable for PCRannealing and extension (typically 50°-75° C.), such as 60° C. bytemperature control system 178. A suitably programmable control systemwhich may be used is disclosed in U.S. Pat. No. 5,038,852 and in U.S.patent application Ser. No. 07/871,264, filed Apr. 20, 1992.

Peltier devices are ideal for controlling the temperatures of the metalblocks because these metal block heat exchangers are each maintained ata constant temperature. Suitable known temperature sensing and feedbackcontrol circuits (not shown) are necessary to control the direction ofcurrent flow through the Peltier devices to maintain the blocktemperature constant by extracting heat from the block when it gets toohot and adding heat when it gets too cold. Any other temperature controlsystem will also work for the blocks such as resistance heaters and/orheated/chilled fluid circulating through passages in the metal blockswith the chilled fluid being, for example, tap water or antifreezechilled by circulating freon of a refrigeration unit, depending upon thedesired temperatures of the blocks. The temperature control systems areshown only once in FIG. 12 and are omitted in subsequent drawingsillustrating the steps of carrying out the PCR process using thedepicted device to avoid unnecessary repetition.

In this embodiment, at least one thin-walled capillary tube reactionchamber 180 runs through the two metal blocks 170 and 172 with a portionof the tube 180 surrounded by the metal block heat exchanger 170 andanother portion surrounded by the metal block heat exchanger 172. Thethin-walled capillary tube can be plastic, metal, or glass, with plasticbeing preferred. Glass can interfere with the PCR reaction by causingthe template strands or the polymerase to stick to the walls, therebyinterfering with the anneal/extend process. Preferably theinterconnection between the capillary tube and the metal block heatexchangers is a slight friction fit, if the tube and block are designedto remain stationary relative to each other, such that the capillarytube 180 can be quickly and easily replaced by sliding a new capillarytube into the metal blocks, thereby rendering the capillary tubesdisposable. This feature minimizes the risk of cross-contamination andmay simplify use of the device by eliminating the need to wash and/orsterilize the capillary tubes between PCR runs. Alternatively, the tubescan be cleaned between PCR runs.

Introduction of a reaction mixture sample and movement within thecapillary tube 180 may be accomplished with a positive displacementpumping device such as a syringe or a peristaltic pump. In addition, anair or liquid buffer may be used between the positive displacementpumping device and the reaction mixture sample.

Introduction of a sample and reaction mixture into tube 180 isaccomplished as shown in FIG. 12 via an automated syringe. A thin rod182 forming a piston 184 on one end thereof is inserted into thecapillary tube 180 through blocks 170 and 172 by a stepper motor 186.The piston 184 forms a seal with the walls of the capillary tube 180.This stepper motor 186 is controlled by a controller such as a suitablyprogrammed computer 188. The function of the stepper motor 186 couldalso be performed by a pneumatic system. One end 190 of the capillarytube 180 is in fluid communication with a reservoir (not shown) ofsample and reaction mixture during mixture introduction. The steppermotor 186 is actuated to withdraw the piston 184 back through thecapillary tube 180, thereby lowering the pressure therein belowatmospheric pressure. This process causes the reaction mixture 192 to bepushed or drawn into the capillary tube 180 until there is enoughreaction mixture in the tube 180 to completely fill the capillary tubevolume within one block as shown in FIGS. 13 and 14.

During denaturation, shown in FIG. 13, the controller 188 and steppermotor 186 position the rod 182 and piston 184 at the left edge of theheat exchanger 170 so that the reaction mixture 192 inside the capillarytube 180 is contained within the heat exchanger 170. The reactionmixture 192 is thus rapidly heated to 94° C. The piston 184 remains inthis position for a time determined by data stored in the computer 188defining the desired time for the denaturation incubation of one cycleof the PCR protocol. These data either can be programmed into thecontroller by the user through a user interface or permanently stored inthe memory of the controller.

The anneal/extend incubation is started when the computer 188 andstepper motor 186 move the rod 182 and piston 184 in the direction topush the reaction mixture 192 to a position in the capillary tube thatis completely surrounded by the heat exchanger 172 as shown in FIG. 14.Here, the reaction mixture is rapidly cooled to a temperature of between50°-70° C. and typically 60° C. to cause the primers to anneal to thesingle-stranded templates and begin formation of the long extensionproduct. At the completion of the anneal/extend incubation, the mixture192 is then ready for a new cycle of denaturation as in FIG. 13 to splitthe double-stranded extension product into single-stranded templates.

The controller and stepper motor then withdraw the rod 184 and piston182 and mixture 192 back into the heat exchanger 170 to begin anotherdenaturation incubation. The cycle of incubations is repeated the numberof times programmed by the user or stored permanently in the database ofthe computer 188.

After the PCR protocol has been completed, the reaction mixture 192 isdischarged into a suitable receptacle as shown in FIG. 15. The pistonpushes the mixture 192 through blocks 170 and 172 and out of thecapillary tube 180 into a container 194.

FIGS. 16-19 illustrate an alternative fifth embodiment of a capillaryPCR instrument similar to that just described except that the functionof the rod 182 and piston 184 are replaced by an inert oil 203. Theinert oil 203 is directed through a sample valve loop 202 from an oilreservoir 204. The oil may be either pumped or driven under pneumaticpressure through the sample valve loop 202 so as to push and pull thePCR reaction mixture back and forth in front of it between the block 170and block 172. This pumping or pneumatic action on the oil is under thecontrol of a computerized controller (not shown) which responds tostored data defining the desired PCR protocol. The valve loop positionand reservoir pressure are computer controlled via a stepper motorinitially to fill completely the capillary tube 180 with oil as shown inFIG. 16. A reaction mixture sample 192 is then pulled into the tube 180as the oil 203 is withdrawn back into the reservoir 204 until thereaction mixture sample 192 is entirely surrounded by the heat exchanger170 as shown in FIG. 17 for the denaturation incubation.

After the denaturation incubation is complete, the oil 203 is againpumped further by the controller so as to push the PCR reaction mixtureto a position in the capillary tube where it is completely surrounded bythe heat exchanger 172 as shown in FIG. 18. There, the mixture israpidly cooled to the anneal/extend temperature. Upon completion of theanneal/extend incubation, the controller (not shown) will reverse thedirection of the inert oil so as to draw the PCR reaction mixture backinto the heat exchanger 170 to start a new cycle. This cycle ofincubations is repeated the number of times specified in the PCRprotocol data stored in the programmable controller. After the requirednumber of cycles is completed, the controller issues a control signal topump the oil far enough through the capillary tube 180 to expel thefinished product into a collection container 194 or a sample detectionsystem coupled to the end of the capillary tube 180 which is not coupledto the sample valve loop 202.

The samples also may be drawn into capillary tubes as above described toa stationary position and then either the capillary tubes or the heatexchange blocks are moved relative to one another to perform the thermalcycling on the reaction mixture sample. In one such embodiment, thetubes and samples remain stationary an d the blocks are translated backand forth. In another, the blocks remain stationary and the tubecontaining the reaction mixture is translated between them. In theseembodiments, there must be relatively low friction between the blocksand the thin capillary tube to facilitate movement. This featuredecreases the thermal conductance between the block and tube andtherefore limits the thermal response and thus increase overall cycletime. Thermal conductance could be maximized in these alternativeembodiments by the use of a thermally conductive grease or otherlubricant such as mineral oil or ethylene glycol polymer.

Metal Block Multiple Capillary Thermal Cycler

A "breadboard" layout of a sixth embodiment of the present invention isillustrated in FIG. 20. The breadboard capillary thermal cyclingapparatus 210 comprises at least 2 metal-block heat exchangers 212 and214 which are, in this preliminary design, fixed to a bed plate 216. Athird heat exchanger 218, shown in dashed lines in FIG. 20, m ay bepositioned i n between heat exchangers 212 and 214 if a protocolrequiring separate annealing and extension temperatures is utilized.Heat exchangers 212 and 214 are separated by an air gap which insulatesthe two heat exchangers from one another. Alternatively, they may beseparated by any suitable insulative layer such as glass, wool, or afoamed polymer. A personal computer 220 communicates input control datavia line 222 to thermostatic controllers 224 and 226. These controllersmaintain each heat exchanger 212 and 214 at a constant temperature foreither denaturation (typically 94° C. or 95° C.) or annealing andextension (between about 37°-65° C. and typically 60° C.). A pluralityof capillary tubes 228 are routed from a sample transfer device 230,through a support clamp 232, through each of the heat exchangers 212 and214, and through a clamp bar 234 on a tube lift assembly 236.

Each of the capillary tubes is made of DuPont Teflon® and preferably hasan internal diameter of about 1.5 mm and a wall thickness of about 0.3mm. This tubing may be obtained from Zeus Industrial Products, Inc. Thetips 238 of the capillary tubes 228 are arranged in a linear rowvertically over a microtiter tray 240 by the tube lift assembly 236. Acomputer-controlled stepper motor 242 rotates lead screws which raiseand lower the clamp bar 234 to in turn raise and lower the tube tips 238into and out of a row of sample containers 244 arranged with tray 240.In this embodiment, twelve capillary tubes 228 are provided to be ableto simultaneously handle one entire row of sample containers 244 inwells in the 96-well microtiter tray 240. However, other numbers ofsimultaneously operating reaction tubes are clearly compatible with theinvention.

The breadboard apparatus 210 uses cartridge heaters embedded in heatexchanger blocks 212 and 214. The temperature controllers 224 and 226for the cartridge heaters are Watlow 922A210 real-time proportionaltemperature controllers, which have RTD or thermocouple temperaturesensors embedded in the thermal cycler blocks 212 and 214 (not shown).Other regulated heat sources could also be used such as surface-mountedelectrical heaters or fluids circulated from constant temperaturereservoirs.

The microtiter tray 240 is typically a plastic tray having an 8 by 12array of wells, each well holding a plastic sample container 244. Thetray 240 is mounted on a movable platen or sliding stage 246, part of atranslation assembly 247. The translation assembly 247 comprises thestage 246, a stepper motor 248 mounted on a base plate 216, and a leadscrew 250. The stepper motor 248 is controlled by the computer 220 andis connected through the lead screw 250 to a fixed arm 252 which isattached to the stage 246. The stepper motor 248 rotates the lead screw250 to index the stage 246 back and forth to various positions under thelift assembly 236. Both the stepper motor 248 and the stepper motor 242are programmably controlled by computer 220 via line 253.

Mounted adjacent to the microtiter tray 240 on the stage 246 is acleaning tray 254. An enlarged partial side view of the stage 246 andtrays 240 and 254 is shown in FIG. 21. The tray 254 has three paralleltroughs, C, R, and W. These troughs contain a cleaner, a rinse liquid,and a waste drain, respectively. The cleaner may be a 10% solution ofchlorine bleach. The rinse solution may be 2.8 Molar sodium thiosulfate,as set forth in Prince, A. M. and Andrus, L., "PCR: How to Kill UnwantedDNA", BioTechniques 12, #3, 358-360 (1992). This tray 254 is used toclean and rinse the capillary tubes 228 before the stage 246 is indexedbetween rows of sample containers 244. The cleaning tray 254 may beconnected to fluid supply reservoirs to keep the cleaner and rinsetroughs full and to a waste container to receive waste discharge fromthe waste drain. The trays 240 and 254 are removably mounted on thesample stage 246. The sample stage 246 is indexed back and forth undercomputer control via line 253 to stepper motor 248 so as to align therow of capillary tube tips 238 above the appropriate trough or samplerow.

The tube lift assembly 236 comprises a drive bar 256 which rides onbearings which turn on a pair of lead screws 258. The lead screws 258are synchronously driven by the computer controlled stepper motor 242 toraise and lower the drive bar 256. The clamp bar 234 is bolted to thedrive bar 256 and includes a bolted plate which clamps the tips 238 ofthe capillary tubes 228 in a vertical orientation over the translatorassembly 247 which horizontally positions the cleaning tray 254 and themicrotiter tray 240.

The sample transfer device 230 in this breadboard design is more clearlyshown with its cover removed in FIG. 22. Sample transfer device 230includes a fixed stage 260 which has up to twelve syringes 262 connectedto the capillary tubes 228 and horizontally mounted and clamped in aspaced array in the fixed stage 260. The cylinders 264 of the syringes262 may be connected to the ends of the capillary tubes 228 individuallyor they may be ganged together. For example, a total of six syringes 262may be used with each syringe feeding two capillary tubes 228.

The plungers 266 are captured between a movable plunger clamping plate268 and clamping bar 270. The clamping plate 268 is mounted on ahorizontally slidable stage 272 driven by a stepper motor 274. Thestepper motor 274 is controlled by computer 220 via line 275 and movesstage 272 back and forth in order to aspirate, translate, and dischargethe PCR reaction mixture sample in each of the capillary tubes 228.Optionally, the syringes may be replaced with one or more peristalticpumps to perform the same function.

This sixth embodiment operates under computer control through atube-cleaning cycle prior to each PCR protocol performed on a row ofsamples. First, the sample stage stepper motor 248 indexes the samplestage 246 to a position at which capillary tube tips 238 are directlyover the cleaner trough C. The lift assembly 236 stepper motor 242 thenlowers the tube tips 238 into trough C. The stepper motor 274 in thesample transfer device 230 is then energized to withdraw the plungers266 to aspirate a slug of cleaner fluid such as bleach into thecapillary tubes 228 to a position past the thermal platens 212 and 214.The lift assembly 236 then raises the tips 238 from the fluid in thetrough C. The sample stage 246 is then moved horizontally via steppermotor 248 to position the waste trough W under the capillary tube tips238. The sample transfer device 230 is then oscillated back and forth tomove the cleaner back and forth in the capillary tubes 228 apredetermined number of times and distances to ensure completecleansing. When this clean cycle is ended, the lift assembly 236 thenlowers tips 238, via stepper motor 242, into the trough "W". At thispoint, the sample transfer device 230 inserts the plungers 266 to expelthe cleaner from the capillary tubes 228 into the waste trough W.

Second, the lift assembly 236 then raises the tube tips 238 out of thewaste trough "W", and the sample stage 246 is then indexed to positionthe tube tips 238 over the rinse trough "R". The lift assembly 236 thenlowers the tips 238 into the rinse trough "R", and the sample transferdevice 230 then aspirates rinsing solution into the capillary tubes 228.The rinsing solution may include a reducing agent such as sodiumthiosulfate to restore tube condition in preparation for the next PCRrun. The tube tips 228 are then raised and the sample transfer device230 again may be oscillated back and forth as required to effectivelyrinse the capillary tubes. The lift assembly 236, transfer device 230,and translator assembly 247 are then operated to position the row ofcapillary tube tips 238 over the waste trough "W" and discharge therinse fluid from the capillary tubes 228 into the waste trough.

Third, the lift assembly 236 and translator assembly 247 are thenoperated under command of computer 220 to position a row of samplecontainers 244 under the capillary tube tips 238. The lift assembly 236then lowers the tube tips 238 into the sample containers 244, and thestepper motor 274 of the sample transfer device 230 withdraws plungers266 aspirating a predetermined quantity of reaction mixture sample intoan initial position within each of the capillary tubes 228. The steppermotor 242 of the lift assembly 236 is then actuated in reverse to liftthe tips 238 out of the row of the sample containers 244.

The stepper motor 274 then continues to withdraw the plungers 266 topull these reaction mixture samples in the capillary tubes 228 throughthe tubes through the thermal heat exchanger 214 to a position in whichthe samples are fully within the heat exchanger 212. The stepper motor274 is operated intermittently to slowly move the samples between thetube tips 238 and the heat exchanger 214. The intermittent movementpermits the fluid at the rear meniscus of the sample slug to catch up tothe main body of the sample slug without forming a separate bubble inthe capillary tube. This effect of surface tension may be minimized bymaintaining the length of capillary tube outside the heat exchangers 212and 214 at elevated temperature. However, since the breadboard apparatus210 described here was operated in a room temperature environment,intermittent movement of motor 274 was required in order to allow thefluid in the meniscus to catch up to the main body of the sample slug.This phenomenon is often called "dragout". Dragout may also be minimizedby minimizing the internal surface roughness of the tubing and/orcoating the interior walls with a polymer such as parylene.

Once the samples reach heat exchanger 214, stepper motor 274 may beoperated continuously to position the samples fully within heatexchanger 212. The first incubation of the PCR protocol begins when thesamples are fully positioned in the heat exchanger 212. The plungers 266are then alternately inserted and withdrawn via stepper motor 274 totranslate the reaction mixture samples between the heat exchangers 212and 214 in accordance with the PCR protocol stored in the computer 220.At the completion of the protocol, the stepper motor 242 of the liftassembly 230 again lowers the capillary tube tips 238 into the samplecontainers 244. The stepper motor 274 then energizes to insert theplungers 266 to discharge the PCR product back into the samplecontainers 244.

The cleaning cycle and thermal cycling steps are then repeated as justdescribed for the next row of sample containers 244 in the microtitertray 240. Operation of the stepper motors in the above described processis programmed into the computer 220 by the user. The steps programmedmay be varied and may include discharge of the PCR product to adifferent row of sample containers 244 than as described above. Theabove sequence is illustrative only.

Optionally mounted between the microtiter tray 240 and the cleansingtray 254 is a capillary tube pressure seal fixture 280. Fixture 280 isshown in FIG. 21 and an enlarged sectional view is shown in FIG. 28.This seal fixture 280 is a plastic or metal block 282 with one row of 12wells 284 which may be aligned beneath the row of capillary tube tips238. Each well 284 may be encircled by an O-ring seal 286 mounted in arecess in the top surface of the block 282 or one continuous O-ring 286may extend around all 12 wells 284. The block 282 is designed to matewith the bottom surface of the clamp bar 234 as shown in FIG. 28 withthe O-ring seal 286 compressed therebetween and the tube tips 238enclosed within the wells 284. The capillary tubes 228 are either pressfitted through a one piece clamp bar 234 or the clamp bar 234 may be atwo piece arrangement with an O-ring seal 288 sandwiched between theupper and lower pieces around each capillary tube 228 as is shown inFIG. 28.

The pressure seal fixture 280 permits the samples in the capillary tubesto be translated in a pressurized environment during the PCR protocol.The fixture is preferably used in situations when the apparatus isoperated at high elevations where the boiling point of the reactionmixture sample is equal to or less than the denaturation temperaturecalled for in the PCR protocol. However, use of this fixture may beavoided by the addition of a denaturant such as formamide to thereaction mixture. The addition of a denaturant lowers the temperaturerequired for denaturation and also slightly raises the boiling point ofthe solution.

If the samples need to be cycled in a pressurized environment duringperformance of the protocol, the sample handling sequence previouslydescribed is slightly modified. Instead of withdrawing a reagent mixsample into the heat exchanger 212 to start the protocol, the sampletransfer device 230 continues to aspirate the sample to a position thatis a predetermined distance beyond the heat exchanger 212. The liftassembly 236 and translator assembly 247 then move to position the tubetips 238 over the fixture 280 and then lower the tips 238 into the wells282 until the O-ring seal 286 is compressed between the fixture 280 andthe underside of the clamping bar 234. In this position, tubes 228 arenow isolated from ambient environmental pressure. The sample transferdevice 230 then pushes the samples in the capillary tubes 228 back intothe heat exchanger 212 for the first denaturation incubation,simultaneously pressurizing the sample volume, the air within thecapillary tubes, and the enclosed air volume beneath the tube tips 238in the sample wells 284 to a minimum pressure above atmospheric. Sampletransfer device 230 is then operated to position the samples alternatelybetween the heat exchangers 212 and 214 to execute the thermal cyclingprotocol in accordance with the instruction data set provided by theuser into the computer 220.

Rotary Thermal Platen Capillary PCR Thermal Cycler

A seventh embodiment 298 of the capillary thermal cycler apparatus inaccordance with the present invention is shown in FIGS. 23 through 27.This embodiment is similar to the sixth embodiment 210 just describedexcept that the heat exchanger blocks 212 and 214 have been replaced bya rotary cylindrical drum heat exchanger assembly 300. Operation of theseventh embodiment 298 is identical to the sixth embodiment justdescribed in the sample handling aspects. The principal difference liesin the operation of the heat exchanger assembly 300. The heat exchangersassembly 300 is moved relative to the capillary tubes 228 in thisembodiment while the samples in the capillary tubes 228 remainstationery.

Like numbers from the sixth embodiment will be used to describe likecomponents in the seventh embodiment in the following description. Asimplified side view of this seventh embodiment is shown in FIG. 23. Aperspective view of the apparatus 298 is shown in FIG. 24.

In this apparatus 298 a microtiter tray 240, a pressurization fixture280, and a cleaning trough plate 254 are translated by a translatorassembly 247 back and forth beneath a capillary tube tip-lift assembly236 under computer control via bus line 253 from personal computer 220as previously described. A sample-transfer device 230 is actuated todraw a reaction mixture sample into the capillary tube tips 238 and theninto a position immediately under the heat exchanger 300. However, inthis embodiment, the sample is not translated back and forth during thePCR protocol. The sample remains stationary and the heat exchangerassembly 300 is rotated to subject the samples to the varioustemperature incubations of the protocol. As in the sixth embodiment, thethermal cycling apparatus 298 of the seventh embodiment utilizes sampletransfer device 230, a ganged syringe assembly, as shown in FIG. 22. Thesample transfer device 230 may also be a peristaltic pumpingarrangement, or a pneumatic pumping device, all automatically controlledby computer 220 via bus line 275. The translation assembly 247 indexesthe microtiter tray 240, cleaning tray 254, and pressurization fixture280 under the row of capillary tube tips 238. The lift assembly 236raises and lowers the capillary tube tips 238 into and out of the samplecontainers 240, troughs C, R, and W, and the sample wells 286 aspreviously described.

The heat exchanger 300 is a cylindrical drum-shaped body divided intofour, axially extending, wedge-shaped segments 302, 303, 304, and 306,each separated from the other by the legs of an "X" shaped radialinsulating layer 308. The drum segments 302 and 306 are utilized for thePCR incubations. Segment 302 is typically maintained at about 60° C. oranother temperature corresponding to the annealing and extensiontemperature by temperature controller 301. Segment 306 is maintained atthe denaturation temperature, typically 95° C., by temperaturecontroller 305. The other two segments 303 and 304 are transitionalsegments. The temperature of these segments will range between 2° C. and20° C. higher or lower than the incubation segments 302 and 306. Segment304 is maintained at a temperature between 2° C. and 20° C. above thedenaturation temperature of approximately 95° C. at which the segment306 is maintained and is typically at about 100° C. maintained bytemperature controller 307. The segment 304 is used to accelerate thetransition, i.e. increase the ramp rate from the annealing/extensiontemperature to the denaturation temperature, to minimize the totalprotocol period. In contrast, the remaining segment 303 is maintained ata temperature significantly below the annealing and extensiontemperature of typically about 60° C. Segment 303 is maintained in arange of between about 2° to 20° C. below the extension temperature andis typically maintained at about 50° C. by the temperature controller309.

The seventh embodiment 298 with four heat exchange segments, 302, 303,304, and 306, is believed to be optimal for a PCR protocol in which asingle denaturation incubation temperature and a singleannealing/extension incubation temperature are used. The third andfourth segments, 303 and 304, kept at a higher and a lower temperature,respectively, than the incubation temperatures, are preferred in orderto maximize efficiently the ramps up and down between theannealing/extension temperatures and the denaturation temperature, asthese transition times should be minimized in order to maximize theproduction of the reaction product in a given time period. It should beunderstood that alternative embodiments are envisioned which have adifferent number of segments, such as two, three, or more segments,depending on the particular protocol required to be performed. Forexample, a five or six segmented heat exchanger could be optimized foreither two- or three-temperature incubation protocols.

The heat-exchange assembly 300 is preferably rotated back and forth tomaintain the temperatures within the required limits in order tominimize the complexity of fluid and/or electrical connections to thesegments. In the breadboard embodiment 298 illustrated, the segments aremaintained at temperature by heaters embedded in each aluminum segment.In addition, an RTD or thermocouple is embedded in each segment toprovide the necessary control signals to the temperature controllers301, 305, 307, and 309. The heat-exchange assembly 300 could,alternatively, be rotated in one direction continuously if suitableslip-ring electrical connections are made to supply heater current andprovide temperature detection and control. This alternative may bepreferable in a production machine to minimize lead wire fatigue.

A side view of the rotating heat exchanger assembly 300 is shown in FIG.26. A rear view of the heat exchanger assembly 300 is shown in FIG. 27.The heat exchanger assembly 300 is horizontally and rotatably supportedfrom the breadboard base plate 216 by two stationary support posts 310.Each support post houses a bearing (not shown) which in turn rotatablysupports an axial shaft 312. The axial shaft 312 has a pulley 314 fixedto one end which is in turn connected via a belt 316 to a stepper motor318 as shown in the end view of FIG. 25. The stepper motor 318 isconnected to and controlled by computer 220. Each of the segments 302,303, 304, and 306 are wedge shaped, solid aluminum segments, withopposite ends bolted to circular end plates 320. The end plates 320 are,in turn, each fixed to the axial shaft 312 which rides in the bearingsheld by the stationary support posts 310.

An indexing disk 322 is also fixed to the shaft 312 adjacent to thepulley 314. This disk is coupled to an optical position sensor 324mounted on the stationary post 310 at the pulley end of the shaft 312.The position sensor 324 is connected through the computer 220 to thestepper drive motor 318. This optical position sensor detects thepresence of a notch 326 in the disk 322 to indicate to the computer 220when the heat exchange drum 300 is correctly positioned.

Each of the segments 302, 303, 304, and 306 preferably has a curvedouter surface 328 with twelve, parallel, semicircular grooves 330therein equally spaced along the length of the segment. Each groove 330is sized to receive one of the capillary tubes 228 so that at least halfof the circumference of the tube 228 is in full contact with the outersurface 328 of the segment. The space between each of the segments ispreferably filled with a thermally insulative material such asfiberglass, plastic, or air. The objective here is to minimize the heattransfer among segments 302, 303, 304, and 306.

Each segment 302, 303, 304, and 306 has an axially extending bore 332therethrough receiving a heater element 334. A single resistance-heaterelement 334 is utilized in each segment in the embodiment 298illustrated. However, dual elements also could be used. Each segmentalso contains a recess for an RTD or thermocouple temperature detectorfor temperature control. In addition or alternatively, each segmentcould have one or more flow channels therethrough, through which aconstant temperature fluid is circulated to maintain the segmenttemperature within the required limits. Each heater element andtemperature detector is connected to its controller 301, 305, 307, or309 via a conventional lead wire 336.

Since the heat-exchanger drum 300 is rotated and the sample ismaintained in a static position during thermal cycling, tensioningdevices 400 and 402 are provided to reduce the wear on the capillarytubes 228 during operation and ensure consistent thermal contact betweenthe tubes and the heat exchanger. Referring now to FIG. 25, each of thetensioning devices 400 and 402 includes an inclined stationary supportblock 404 mounted on bed plate 216 and a sliding block 406 and cap 408bolted to the block 406 which clamps the capillary tubes 228 in parallelpositions. The blocks 406 and caps 408 position the capillary tubes 228along a tangent line to the grooves 330 in the surface 328 of thesegments of the heat exchanger assembly 300. Each of the sliding blocks406 is spring-biased away from the heat exchanger 300 in the tangentialdirection by coil springs 410. A pair of retaining springs 412 actingnormal to the surface of the blocks 406 are used to retain the each ofthe sliding blocks 406 against the inclined upper surface of each of thestationary support blocks 404.

The tensioning devices 400 and 402 are positioned on the bed plate 216so that each of the capillary tubes 228 contacts the entire groovelength of the curved outer surface 328 of each particular segment 302,303, 304, and 306 when the respective segment is positioned at a "sixo'clock" position as is shown in FIGS. 24 and 25. The springs 410 and412 cooperate to ensure that the portions of the capillary tubes 228under the heat exchanger 300 are maintained in good thermal contact withthe outer surface 328 of each segment and that each tube is retained inits groove 330.

In order to reduce wear on the tensioned capillary tubes 228, a tensionrelease mechanism 420, driven by a stepper motor 422, automaticallyreleases the tension on the tubes 228 during rotation between theheat-exchanger segments. A horizontally mounted rotating shaft 424,driven by motor 422 via computer 220 through bus 423, positions aneccentric lobe 426 against the sliding block 406, in opposition to thespring bias of spring 410. The eccentric lobe 426 pushes the slidingblock 406 toward the heat exchanger 300 to release tension on thecapillary tubes 228 slightly before or simultaneously with a drivesignal transmitted from the computer 220 to the heat exchanger steppermotor 318.

An optically transparent thermal shield 340 may be positioned beneathand closely adjacent to the segment in contact with the capillary tubes228. This thermal shield 340 may also act as a window to a fluorescencedetector 350 mounted under the heat exchanger 300 as shown in FIGS. 26and 30 to monitor the production of reaction product during the DNAamplification process.

Ethidium bromide, an intercalating dye, is dissolved in the reactionmixture at a level which does not interfere with the PCR. Ethidium ionsfluoresce at about 615 nm with a higher intensity when intercalated inthe DNA than when free in solution. Consequently, the intensity offluorescence may be used as a measure of the concentration of theamplified DNA population.

The detector 350 uses a photodiode array (PDA) which detectsfluorescence from all twelve of the capillary tubes 228 simultaneouslyduring incubations. The detector 350 is schematically shown in FIG. 30.Mounted beneath the drum 300 and between tensioning devices 400 and 402is a housing 352 which contains and supports an ultraviolet light source354 and a mirror 356. The UV source 354 is directed toward the portionsof the twelve capillary tubes in contact with the drum 300. The mirroris positioned so that light emitted by or reflected from the capillarytubes under the drum 300 will be directed through an aperture 358 in thehousing 352 and through a focusing lens 360 onto a photodiode array 362.The output of the photodiode array 362 is fed through conventionalamplifier circuitry 364 to the computer 220 for display.

In this breadboard design, light from each of the 12 capillary tubes isdirected to a separate photodiode in the array so that amplification ineach tube can be simultaneously and individually monitored. The presentdiode array used consists of 35 diodes in a linear array. Every thirddiode is optically coupled as a signal diode to one of the capillarytubes 228. An adjacent diode to each signal diode is used to monitor andelectronically subtract background away from the gross signal out ofeach signal diode.

The annealing/extension section of the drum 300 is positioned againstthe capillary tubes 228 during monitoring. The detected fluorescenceintensity provides real-time monitoring of the growth of PCR productduring the PCR protocol. This information also can be used to estimatethe amount of target DNA originally present prior to amplification, as,during most of the amplification, the growth rate is a simpleexponential process.

The interior of the housing 352 is painted flat black to absorbscattered light and is closely fitted adjacent the bottom of the drum300 to exclude ambient light. The outer surface 328 of theannealing/extension segment 302 is also painted flat black to absorbscattered light. The curved surface of each of the grooves 330 in theannealing/extension segment 302 is polished to a mirror finish so as toreflect the maximum amount of the fluorescence.

Alternatively, other segments could also be used for detection. However,it is felt that the long duration of the annealing/extension incubationprovides the optimal opportunity for monitoring the PCR product buildupfrom cycle to cycle. In the detector system 350, fluorescence data arereceived once each second during the annealing/extension incubation. Inaddition, the entire detection system would preferably be self containedin a production instrument.

The detection system just described may be adapted to many of theembodiments herein described so long as the capillary tubes arerelatively transparent so as to pass UV and visible light. In addition,the detection system signal may be used as an input to the computer tocalculate and adjust optimum incubation times. Alternatively, thedetector output may be used to provide a termination signal for the PCRprotocol. In other words, a desired quantity of PCR product could bespecified to the computer 220 rather than a total number of cycles as iscurrently the practice in the art. Finally, other detection systems maybe utilized. For example, CCD devices may be used instead of a diodearray for enhanced resolution and sensitivity. The detector also may becoupled to a polychromator, and several fluorophores with differentemission spectra can be used to distinguish among various components ofthe reaction mixture. For example, one fluorescent signal, generated bya dye which does not interact with the DNA amplification, can be used torecalibrate the fluorescence optics continuously, compensating forair-bubble formation or solute concentration consequential to solventevaporation. The optical calibration dye should resist photo-bleachingor, if the DNA-reporting dye photo-bleaches, be chosen to show similarphotosensitivity. A second fluorescent signal can be provided by a dyewhich experiences a strong fluorescence enhancement upon binding toduplex DNA, such as ethidium, the intercalating dye well known in theart of nucleic acid analysis, or a member of the bis-benzimide class ofdyes, known to bind to the minor groove of duplex DNA. Higuchi et al,"Simultaneous Amplification and Detection of Specific DNA Sequences,"Biotechnology 10, Apr. 1992, pp. 413-417, showed that ethidium could beused in the PCR mixture to monitor the accumulation of duplex DNA inreal time. Alternatively to this second fluorescent signal, one mayattach a dye and a fluorescence quencher to a probe which anneals to PCRtemplate, in such a way that when PCR is performed with a DNA polymerasepossessing 5'→3' exonuclease activity, the nick translating activity ofthe polymerase digests the probe. As disclosed by Holland et al, PCTPubl. No. WO 92/02638!, this digestion activity would generate afluorescent signal as the dye is split away from the quencher. Use ofseveral, spectrally distinct, fluorophore-quencher pairs attached toprobes specific for different templates would allow simultaneousmonitoring of accumulation of several PCR products.

The apparatus 298 operates automatically as follows, under control ofcomputer 220. A reaction-mixture sample is drawn into each of thecapillary tubes 228 via the lift assembly 236 and sample-transfer device230 as previously described. The sample is aspirated from the samplecontainers 244 into the capillary tubes 228 to a position directly underthe heat exchanger drum 300. As shown in FIG. 24, segment 306 contactsthe section of capillary tube 228 containing the samples. Thetemperature of the samples in the capillary tubes 228 is quickly raisedup to denaturation temperature of 95° C. The denaturation temperature ismaintained in the capillary tubes 228 for the required incubationperiod.

At the end of this period, the assembly 300 is rotated 90° clockwise toposition the segment 303 in contact with the capillary tube sectioncontaining the reaction-mixture samples. Because the segment 303 ismaintained at about 50° C., the samples rapidly cool to a temperature ofabout 60° C. At an appropriate time, the assembly 300 is rotated another90° clockwise to position segment 302 against the capillary tubeportions containing the samples. The segment 302 is maintained at 60° C.The assembly remains in this position again for the required incubationtime dictated by the PCR protocol. At the end of the annealing andextension incubation, the drum is rotated clockwise another 90° so thatsegment 304 (which has a temperature typically 100° C.) is in contactwith capillary tubes 228 for the time necessary to ramp the reaction mixtemperature quickly to about 95° C. Upon reaching 95° C., the heatexchanger assembly 300 is rotated quickly counterclockwise another 270°to place segment 306 again in contact with the capillary tubes 228 forthe next required denaturation incubation. This process is repeated asrequired to complete the PCR protocol. During each period of rotation,the cam eccentric 426 is rotated by motor 422 to detension the capillarytubes 228 from the outer surface 328 of the segments. When rotationceases, the eccentric 426 is rotated to reengage the tensioning devices400 and 402 to ensure adequate thermal contact during the incubations.

As in the sixth embodiment described above, rotation coordination andcontrol of the heat exchanger assembly and temperature control of thesegments is maintained by the computer 220. At the end of the PCRprotocol, the computer 220 directs the sample-transfer device 220 toinsert the plungers 266 to discharge the reaction product back into thesample containers 244 in the wells in the microtiter tray 240. Thecleaning and rinse sequence of steps, described with reference to thesixth embodiment, is then performed to flush the capillary tubes of anycontaminants and remnants of the sample just processed. The translatorassembly 247 then repositions the microtiter tray 240 to the next row ofwells holding sample containers 244, and the entire process is repeated.

The capillary tube tips 238 in the sixth and seventh embodiments abovedescribed have a special physical configuration as shown in the enlargedview of a tip 238 in FIG. 29. If the capillary tubes are simply cut offtransverse to the axis of the tube, the last drop of sample beingdischarged often hangs off the end of the tube after dispensing. Thus,complete discharge of the reaction product into the sample well isprevented. It has been found that by tapering the tip wall 290 as shownin FIG. 29, the last drop of reaction product is reliably dischargedfrom the ends 238 of the capillary tube 228.

"Dragout" of the sample in the capillary tubes 228, as the sample ismoved back and forth between heat exchangers in the sixth embodiment,and into position under the heat exchanger drum 300 in the seventhembodiment, is minimized in teflon tubing having minimal internalsurface rough a surface tension phenomenon in capillary tubing where thefluid adjacent the wall tends to adhere progressively to the wall,creating a generally parabolic longitudinal cross sectional shape to theends of the sample slug. If the dragout is severe, the trailing end canseparate or "drag out" from the main body of the sample slug during slugmovement, coalesce, and eventually trap an air bubble between endportions of the sample, thus extending the overall length of the sample.As previously mentioned, the ambient temperature conditions of thesamples in the capillary tubes contribute to dragout. Accordingly,maintaining an elevated temperature of the sample and the capillary tubeduring aspiration into the capillary tubes may minimize dragout. Anothereffective way to minimize dragout is to break the process of aspiratingthe sample into position into many discrete steps and pauses. Thesepauses permit the trailing end of the sample to catch up to the mainsample body in the capillary tube.

There is an alternative to using a pressurization fixture 280 atelevations such as Denver, Colo., where the boiling point of water isreduced to 93.5° C. This alternative is the addition of a denaturantsuch as formamide in the reaction mixture. This approach is suggested inPanaddio et al, "FoLT PCR: A Simple PCR Protocol for Amplifying DNADirectly from Whole Blood," BioTechniques Vol. 14, No. 2 (1993) pp.238-243. The authors used thermostable Tth polymerase rather than Taqpolymerase and showed that a 95°-50°-70° C. protocol could be convertedto an 85°-40°-60° C. protocol for a reaction mixture containing 18%formamide.

Taq polymerase tolerates a number of DNA denaturants including glyceroland acetamide. In fact, 2.5% formamide in the reaction mixture samplesutilized in the seventh embodiment of the invention described above hasyielded satisfactory amplification at a denaturation temperature of 89°and a annealing/extension temperature of 63°, thus lowering the protocoltemperatures uniformly by about 5° C.

The following paragraphs refer to FIGS. 31-34 which provide a flow chartof the software utilized to control the operation of the seventhembodiment of the present invention. The software in computer 220controls the five stepper motors. These stepper motors are: motor 274,which operates the syringes 262 in pump 230; stepper motor 318, whichdrives the heat exchange drum 300; stepper motor 248, which translatesthe sample stage 246 horizontally; the stepper motor 242, whichvertically raises and lowers the tips 238 of the capillary tubes 228;and stepper motor 422, which rotates cam 426 to tension and detensioncapillary tubes 228 against the heat exchange drum 300.

The software may be programmed in Basic or any other conventionalprogramming language known to those skilled in the art. The presentbreadboard design described below is programmed in Basic.

Referring now to FIG. 31, the user starts at block 501, and loads therequired variables for the thermal cycling protocol. These variablesinclude the denaturation temperature, annealing temperature, incubationtime at the denaturation temperature, incubation time at the annealingtemperature, number of cycles in thermal cycle A, B, and C, samplevolume, overshoot incubation time, undershoot incubation time, theabsolute pump location which correctly places the midpoint of thesamples at the 6 o'clock position beneath the heat exchanger drum 300,the pump speed for load and the pump speed for unload, and the detectiontime. If all these variables have been entered without error, thesoftware proceeds to block 502, and the detector port is opened to thecomputer 220.

Processing then transfers to the sample load subroutine 503 shown inFIG. 32. This subroutine 503 begins with stepper motor 242 beingenergized to raise the lift tube tips 238 to a position at least about acentimeter above the tubes in the microtiter tray 240. In block 505, thesyringe pump 230 speed is then set. The speed of stepper motor 274 isset to withdraw the ganged syringes 262 at a predetermined rate tominimize dragout. Stepper motor 274 is actuated to close the syringepump 230 in block 506. This action fully inserts the syringe plungers266.

The stepper motor 248 then is energized in block 507 via bus 253 toposition the microtiter plate 240 such that the first of a predeterminedrow of sample tubes or containers is positioned directly under the tubetips 238. Next, in block 508, syringe pump 230 is cocked. Here, thestepper motor 274 moves in the reverse direction to withdraw theplungers 266 to a slightly withdrawn position to ensure that there is avolume of air within the syringe which can be used to expel the samplescompletely from the tubes 228 at the end of the PCR protocol.

Computer 220 then sends a signal via bus 222 to stepper motor 242 tolower the tube tips 238 in block 509. The capillary tube tips 238 arelowered into the sample tubes almost to the bottom of the tubes. Thisstep positions the tube tips so that a maximum volume of sample reactionmix may be withdrawn into the capillary tubes 228.

The syringe pump 230 is then actuated to withdraw preferably a 50microliter sample, in block 510, into the capillary tubes 228. When therequired volume has been withdrawn by the syringe, stepper motor 242 isthen energized in block 511 to lift the tube tips 238 out of the sampletubes. This operation completes the load subroutine 503 shown in FIG.32.

Referring back to FIG. 31, control returns to block 503 and proceeds toblock 512. In block 512, stepper motor 422 is actuated to release thetension between capillary tubes 228 and the drum 300. More particularly,the stepper motor 422 rotates cam 426 to move the sliding block 406toward the thermal cycling drum 300 a few millimeters, which issufficient to detension the capillary tubes. Once the tension isreleased, in block 513, the thermal cycling drum 300 is rotated bystepper motor 318 to a position in which segment 303, which is thelowest temperature, is symmetrically positioned at the 6 o'clockposition. In this position, the capillary tubes 228 fully cover thegrooves 330 in this segment.

As soon as drum 300 positions segment 303 correctly at the 6 o'clockposition, control proceeds to block 514. Here, stepper motor 422 isactuated again to rotate the cam 426 further out of engagement with thesliding block 406, allowing spring 410 to tension the tubes 228 againstthe surface of grooves 330 in this segment of the drum at the 6 o'clockposition. The stepper motor 274 is then intermittently energized, inblock 515, in a "slow load" fashion to move the sample from just abovethe capillary tube tips 238 to a position within the capillary tubesdirectly adjacent the segment 303 at the 6 o'clock position. Thissequence of energizing stepper motor 274 to withdraw the plungers 266 asmall amount and then wait for a predetermined amount of time, typicallya few seconds, moves the sample in a series of forty or fifty shortsteps, and typically takes about two minutes to move the samples intoapproximate position beneath the drum 300. This intermittent motion isnecessary to prevent the "dragout" phenomenon from causing the rear endof the sample volume to separate and form a bubble. Such a bubble wouldeffectively lengthen the sample volume which could impair amplificationof the nucleic acid in the sample. This slow load procedure may not beneeded if the inside surface of the capillary tubes is coated withparylene or the length of capillary tubing is preheated.

Finally, in block 516, stepper motor 274 is actuated to position thesamples precisely and symmetrically under the segment 303.

Referring back to FIG. 3, the thermal cycling subroutines are thenperformed. The thermal cycling protocol is typically 20 to 30 thermalcycles consisting of alternating denaturation incubations andannealing/extension incubations. In this breadboard design, the protocolis broken down into three sequences A, B, and C which are identicalexcept for the number of cycles in each sequence. The block diagram ofthe thermal cycling subroutine shown in FIG. 33 is used for each of thesequences. First, the number of cycles to be performed in thermal cycleA is set in counter N, block 519. The tension on the capillary tubes isreleased in block 520 as above described by energizing stepper motor426. Drum 300 is then rotated clockwise via stepper motor 318 toposition segment 304 at the 6 o'clock position, block 521. Since thedrum 300 is initially positioned with segment 303 at the 6 o'clockposition, the first cycle rotation will be 180° clockwise. In subsequentcycles, rotation in block 521 will be 90° clockwise from theannealing/extension incubation position in which segment 302 is at the 6o'clock position. The stepper motor 422 is again energized to rotate cam426 out of engagement with sliding block 406 to retension capillarytubes 228 in block 522. Block 523 represents the required wait time withsegment 304 in contact with the capillary tubes to quickly ramp thesample temperature up to the 95° C. denaturation temperature. Thisperiod is relatively short, on the order of a few seconds. The greaterthe temperature of the segment 304, the shorter the time required.

After the required wait period, in block 524, tension on the capillarytubes 228 is released. Drum 300 is then rotated clockwise 90°, in block525, to position segment 306 at the 6 o'clock position. In block 526,stepper motor 422 is again energized to rotate the cam 426 and releasethe sliding block 406 and restore tension between capillary tubes 228and the bottom of drum 300. This step begins the denaturation incubationsymbolized in block 527. This denaturation incubation is typically onthe order of one minute. At the end of this incubation, in block 528,stepper motor 422 again energizes to release the tension between thecapillary tubes 228 and the drum 300. Drum 300 is then rotated 270°counterclockwise to return the cold segment 303 to the 6 o'clockposition, in block 529.

The sequential clockwise rotation to 270° and return in acounterclockwise direction prevents the lead wires from the temperaturecontrollers from becoming wound around the drum 300. When the drum 300reaches the position in which segment 303 is at the 6 o'clock position,tension is again restored in block 530. Contact with the drum segment303 drives the temperature of the sample quickly down to theannealing/extension temperature of 60° C. Accordingly, the wait, inblock 531, is relatively short. Again, after the wait in block 531, thetension on the tubes is released, in block 532, and drum 300 is rotatedclockwise 90° to position segment 302 at the 6 o'clock position. Inblock 534, tension is again restored between the tubes 228 and the drum300. In block 535, fluorescence detection is begun and continues duringthe annealing/extension incubation for a period typically about 119seconds. The incubation for annealing and extension is carried out inblock 536. The annealing/extension incubation period and the detectionperiod are about the same.

At the end of the annealing/extension incubation, tension is againreleased in block 537 between the tubes 228 and the drum 300 as abovedescribed. The counter is decremented in block 538 and queried for anull in block 539. If the counter is equal to 0, the subroutine returnsto the main program, block 540. If the counter is not 0, control isdirected back to block 520.

In block 540, pump 230 is actuated to push the sample back and forth andback again so that bubbles are expelled from the sample. During thecontinuous thermal cycling between the denaturation andannealing/extension temperatures, the sample may produce small bubbleswhich cause the ends of the samples to expand or move away from eachother, thus moving the sample volume partially out from beneath thesegment. Moving the sample back and forth by 25 microliters, 50microliters, and then back 50 microliters has been shown to expel thesebubbles from the sample, thus restoring the sample volume to its initialdimensions. Once the bubble sweep block 540 is completed, the thermalcycle B sequence is commenced as shown in block 541. Thermal cycle B isidentical in operational steps to the thermal cycle A above described.Again, after the thermal cycle B, any bubbles are then swept by movingthe sample back and forth in a "bubble sweep", block 542.

Typically, thermal cycle A consists of about five cycles and thermalcycle B about 10 cycles. Thermal cycle C, as shown in block 543,comprises the remaining cycles in the protocol, typically between 15 and20, for a total of 30 to 35 cycles in the protocol. After the completionof thermal cycle C, tension between the tubes 228 and the thermalcycling drum 300 is released as indicated in block 544, and drum 300rotated, if necessary, to position block 303 at the 6 o'clock positionas in block 545.

The sample unload subroutine is then performed in block 546. Thissubroutine is shown in FIG. 34. Initially, the capillary tube tips 238,during the thermal cycling, are in the raised position. First, themicrotiter tray 240 is indexed to the next row or other row asprogrammed by the user, block 547. In blocks 548 and 549, a command isgiven to lift the capillary tube tips to the upper position and set thepump speed (stepper motor 274). The pumping speed for unload is fasterthan in the load subroutine because absolute positioning and dragouteffects are not critical. In addition, in block 550, backlash issuspended. Backlash is a correction factor applied to stepper motor 274to account for various tolerances which accumulate during thealternating insertion and withdrawal of the syringes. In the unloadsubroutine, the end position of the sample is going to be dischargedfrom the capillary tubes. Consequently, absolute position is notimportant. Therefore, compensation for backlash in the pumping assemblyis not required in this subroutine.

In block 551, the pump 230 (stepper motor 274) is actuated to move thesample approximately 20 millimeters from its current position at the 6o'clock position. In block 552, the sample is then moved via pump 230 ina slow "unload" procedure, similar to the load sequence in order toprevent dragout from forming bubbles in the capillary tubing. In theslow unload procedure, the pump moves the sample approximately 8millimeters and then waits for one second. Pump 230 then again actuatesto move another 8 millimeters distance within the capillary until thesample is discharged into the sample tubes in the microtiter tray 240.Finally, in block 553, the syringe pump 230 is fully closed to push thelast remaining amount of sample from the tube tips 238 into the sampletubes with the slug of air from initially cocking the syringes in block508 of the sample load subroutine shown in FIG. 32.

Finally, in block 554, backlash is restored and, in block 555, thedetector port is closed. In block 556, the program looks for a link toanother sample run. If another sample run is required, a cleaningsubroutine is performed, which is symbolized by block 557. This cleaningsubroutine simply involves positioning the microtiter tray transferstage 247 over the appropriate clean, rinse, or waste troughs, loweringthe tube tips 238 into the cleaning tray, and aspirating a slug ofcleaning solution into the capillary tubes 228 to a position beyond theheat exchanger drum 300, and oscillating the pump 230 back and forth toturbulently scrub the interior surfaces of the capillary tubes 228. Thecleaning solution is then expelled into the waste trough. A rinsesolution is next aspirated into the capillary tubes, oscillated back andforth, and then discharged. Finally, a conditioning solution may beaspirated into the capillary tubes in preparation for the next cycle.After this conditioning solution is discharged, the software controlreturns to block 502 for the next row of samples to be thermally cycled.

While the above description is illustrative of the preferred embodimentsof the present invention, it will be appreciated that the inventiveconcept of the capillary thermal cycling apparatus in accordance withthe invention may be practiced otherwise than as specifically described.For example, the various embodiments of the thermal cycling apparatus inaccordance with the invention are applicable to any other nucleic acidamplification and/or reaction techniques besides PCR which requiresthermal cycling at least once. The above examples are illustrative only.

These techniques include ligase chain reaction and repair chain reactionas discussed in Abramson et al., "Nucleic Acid AmplificationTechnologies," Current Opinion in Biotechnology, 1993, Vol 4, pp. 41-47.The ligase chain reaction is also discussed by Francis Barany in "TheLigase Chain Reaction in a PCR World", PCR Methods and Applications1:5-16 (1991). Other methods for which the invention may be used includethe 3SR method discussed by Fahy et al., "Self-sustained SequenceReplication (3SR): An Isothermal Transcription-based AmplificationSystem Alternative to PCR", PCR Methods and Applications 1:25-33 (1991)and the Strand Displacement Assay (SDA) discussed by Walker et al. in"Isothermal In Vitro Amplification of DNA by a Restriction Enzyme/DNAPolymerase System", Proc. Natl. Acad. Sci. U.S.A. 89:392-396 (1992).

These reactions all involve reaction mixtures which undergodenaturation, annealing and extension processes. They primarily differonly in the specific extension mechanisms employed in the primerextension process in which the annealed oligonucleotides are extended toreplicate the target strand. Repair chain reaction and LCR involverepetitive thermal cycling. 3SR and SDA methods involve an initialdenaturation step followed by an isothermal incubation for the annealingand extension processes.

Other potential applications of the above described instruments also mayinclude cDNA synthesis prior to PCR, ligation and kinasing of DNA, andsuccessive enzyme treatments in which reagent additions may be requiredduring incubations or thermal cycling. Thus, the embodiments of theinvention are subject to modification, variation, and change withoutdeparting from the proper scope and fair meaning of the appended claims.Accordingly, it is intended to embrace all such changes, modifications,and variations that fall within the spirit and broad scope of theappended claims. All patent applications, patents and other publicationscited herein are incorporated by reference in their entirety.

What is claimed is:
 1. An apparatus for performing a nucleic acidamplification reaction in a reaction mixture in a capillary tube, thenucleic acid amplification reaction including denaturation, annealingand extension processes, the apparatus comprising:(a) a first heatexchanger including a thermoregulating system for stabilizing thetemperature of the first heat exchanger at a temperature in a range oftemperatures suitable to cause the denaturation process to occur in thereaction mixture; (b) a second heat exchanger including athermoregulating system for stabilizing the temperature of the secondheat exchanger at a temperature in a range of temperatures suitable forcausing the annealing and extension processes to occur in the reactionmixture; (c) the capillary tube being routed so as to have a firstportion in thermal contact with the first heat exchanger and a secondportion in thermal contact with the second heat exchanger; (d) a valveassembly having an input port coupled to the capillary tube forreceiving said reaction mixture into the tube and an output port fordelivering a finished reaction product from the tube, the valve assemblyallowing injection of new reaction mixture in a first position, allowingthe reaction mixture to circulate in the capillary tube between thefirst and second heat exchangers in a second position, and allowingejection of finished reaction product in a third position; and (e) apumping device fixedly disposed in one of the heat exchangers coupled tothe capillary tube so as to circulate the reaction mixture in thecapillary tube such that the reaction mixture passes sequentially andcyclically through the first and second heat exchangers.
 2. Theapparatus of claim 1 further comprising a spectrophotometric detectoroptically coupled to the capillary tube for generating data representingchanges in the reaction mixture during the nucleic acid amplificationreaction.
 3. The apparatus of claim 1 further comprising a valve meanscoupled to the capillary tube for providing an entry port through whichreagents, additional primer, or additional enzyme may be added to thereaction mixture in the capillary tube.
 4. The apparatus of claim 1further comprising a means for injecting a discontinuity adjacent to thereaction mixture in the capillary tube.
 5. The apparatus of claim 4further comprising a cycle counter coupled to the capillary tube forcounting the passage of the discontinuity.
 6. The apparatus of claim 1further comprising a computer-directed controller coupled to the pumpingdevice and to the valve assembly for generating control signals to thepumping device and the valve assembly to automatically implement anucleic acid amplification reaction protocol in accordance with datastored in the computer-directed controller.
 7. The apparatus of claim 6wherein the computer directed controller is user-programmable such thatdata defining different nucleic acid amplification reaction protocolsmay be entered by a user.
 8. An apparatus for performing a nucleic acidamplification reaction in a reaction mixture in a capillary tube, thenucleic acid amplification reaction including denaturation, annealingand extension processes, the apparatus comprising:(a) a first heatexchanger including a thermoregulating system for stabilizing thetemperature of the first heat exchanger at a temperature in a range oftemperatures suitable to cause the denaturation process to occur in thereaction mixture; (b) a second heat exchanger including athermoregulating system for stabilizing the temperature of the secondheat exchanger at a temperature in a range of temperatures suitable forcausing the annealing and extension processes to occur in the reactionmixture; (c) the capillary tube being routed so as to have a firstportion in thermal contact with the first heat exchanger and a secondportion in thermal contact with the second heat exchanger; (d) a valveassembly having an input port coupled to the capillary tube forreceiving said reaction mixture into the tube and an output port fordelivering a finished reaction product from the tube, the valve assemblyallowing injection of new reaction mixture in a first position, allowingthe reaction mixture to circulate in the capillary tube between thefirst and second heat exchangers in a second position, and allowingejection of finished reaction product in a third position; (e) a pumpingdevice in one of the heat exchangers coupled to the capillary tube so asto circulate the reaction mixture in the capillary tube such that thereaction mixture passes sequentially and cyclically through the firstand second heat exchangers; and (f) a spectrophotometric detectoroptically coupled to the capillary tube for generating data representingchanges in the reaction mixture during nucleic acid amplificationreaction.
 9. The apparatus of claim 8 further comprising a valve meanscoupled to the capillary tube for providing an entry port through whichreagents, additional primer, or additional enzyme may be added to thereaction mixture in the capillary tube.
 10. The apparatus of claim 8further comprising a means for injecting a discontinuity adjacent to thereaction mixture in the capillary tube.
 11. The apparatus of claim 10further comprising a cycle counter coupled to the capillary tube forcounting the passage of the discontinuity.
 12. The apparatus of claim 8further comprising a computer-directed controller coupled to the pumpingdevice and to the valve assembly for generating control signals to thepumping device and the valve assembly to automatically implement anucleic acid amplification reaction protocol in accordance with datastored in the computer-directed controller.
 13. The apparatus of claim12 wherein the computer directed controller is user-programmable suchthat data defining different nucleic acid amplification reactionprotocols may be entered by a user.
 14. An apparatus for performing anucleic acid amplification reaction in a reaction mixture in a capillarytube, the nucleic acid amplification reaction including denaturation,annealing and extension processes, the apparatus comprising:(a) a firstheat exchanger including a thermoregulating system for stabilizing thetemperature of the first heat exchanger at a temperature in a range oftemperatures suitable to cause the denaturation process to occur in thereaction mixture; (b) a second heat exchanger including athermoregulating system for stabilizing the temperature of the secondheat exchanger at a temperature in a range of temperatures suitable forcausing the annealing and extension processes to occur in the reactionmixture; (c) the capillary tube being routed so as to have a firstportion in thermal contact with the first heat exchanger and a secondportion in thermal contact with the second heat exchanger; (d) a valveassembly having an input port coupled to the capillary tube forreceiving said reaction mixture into the tube and an output port fordelivering a finished reaction product from the tube, the valve assemblyallowing injection of new reaction mixture in a first position, allowingthe reaction mixture to circulate in the capillary tube between thefirst and second heat exchangers in a second position, and allowingejection of finished reaction product in a third position; (e) a pumpingdevice in one of the heat exchangers coupled to the capillary tube so asto circulate the reaction mixture in the capillary tube such that thereaction mixture passes sequentially and cyclically through the firstand second heat exchangers; (f) means for injecting a discontinuityadjacent to the reaction mixture in the capillary tube; and (g) a cyclecounter coupled to said capillary tube for counting the passage of saiddiscontinuity.
 15. The apparatus of claim 14 further comprising a valvemeans coupled to the capillary tube for providing an entry port throughwhich reagents, additional primer, or additional enzyme may be added tothe reaction mixture in the capillary tube.
 16. The apparatus of claim14 further comprising a computer-directed controller coupled to thepumping device and to the valve assembly for generating control signalsto the pumping device and the valve assembly to automatically implementa nucleic acid amplification reaction protocol in accordance with datastored in the computer-directed controller.
 17. The apparatus of claim16 wherein the computer directed controller is user-programmable suchthat data defining different nucleic acid amplification reactionprotocols may be entered by a user.
 18. The apparatus of claim 14wherein said cycle counter is fixedly disposed in one of said heatexchanges.
 19. The apparatus of claim 14 wherein said cycle counter is aphoto-electric sensor.
 20. The apparatus of claim 14 wherein said cyclecounter is a capacitance sensor.